1-[2-(2,4 dimethylphenylsulfanyl)-phenyl]piperazine as a compound with combined serotonin reuptake, 5-HT3 and 5-HT1a activity for the treatment of cognitive impairment

ABSTRACT

This disclosure relates to a method of treating a disease selected from the group consisting of affective disorders, depression, major depressive disorder, anxiety, general anxiety disorder, social anxiety disorder, obsessive compulsive disorder, panic disorder, and panic attacks. The method includes administering a therapeutically effective amount of Compound I or a pharmaceutically acceptable salt thereof to a patient in need thereof, in which Compound I is 1-[2-(2,4-dimethylphenylsulfanyl)-phenyl]piperazine.

CROSS REFERENCE TO PRIOR APPLICATIONS

This is a continuation of U.S. application Ser. No. 14/242,337, filedApr. 1, 2014, which is a divisional application of U.S. application Ser.No. 12/301,061, filed Nov. 17, 2008, which is a U.S. National Phaseapplication under 35 U.S.C. §371 of International Patent Application No.PCT/DK2007/050075, filed Jun. 15, 2007, and claims the benefit of DanishPatent Application No. PA 2006 00824, filed Jun. 16, 2006; U.S.Provisional Application No. 60/805,014, filed Jun. 16, 2006; DanishPatent Application No. PA 2006 01223, filed Sep. 22, 2006; U.S.Provisional Application No. 60/826,666, filed Sep. 22, 2006; DanishPatent Application No. PA 2006 01384, filed Oct. 25, 2006; U.S.Provisional Application No. 60/862,826, filed Oct. 25, 2006; and DanishPatent Application No. PA 2007 00427, filed Mar. 20, 2007, all of whichare incorporated by reference herein. The International Applicationpublished in English on Dec. 21, 2007 as WO 2007/144005 under PCTArticle 21(2).

FIELD OF THE INVENTION

The present invention relates to compounds, which exhibit serotoninreuptake inhibition activity combined with an activity on the serotoninreceptor 1A (5-HT_(1A)) and the serotonin receptor 3 (5-HT₃), and whichas such are useful in treatment of CNS related diseases.

BACKGROUND OF THE INVENTION

Selective serotonin reuptake inhibitors (SSRI) have for years been thefirst choice therapeutics for the treatment of certain CNS relateddiseases, in particular depression, anxiety and social phobias becausethey are effective, well tolerated and have a favourable safety profileas compared to previously used compounds, i.e. the classical tri-cycliccompounds.

Nonetheless, therapeutic treatment using SSRI is hampered by asignificant fraction of non-responders, i.e. patients who do not respondor only respond to a limited extend to the SSRI treatment. Moreover,typically an SSRI treatment does not begin to show an effect until afterseveral weeks of treatment.

In order to circumvent some of these shortcomings of SSRI treatment,psychiatrists sometimes make use of augmentation strategies.Augmentation of antidepressants may be achieved e.g. by combination withmood stabilisers, such as lithium carbonate or triiodothyronin, or bythe parallel use of electroshock.

It is known that a combination of inhibition of the serotonintransporter (SERT) with an activity on one or more serotonin receptorsmay be beneficial. It has previously been found that the combination ofa serotonin reuptake inhibitor with a compound having 5-HT_(2C)antagonistic or inverse agonistic effect (compounds having a negativeefficacy at the 5-HT_(2C) receptor) provides a considerable increase inthe level of 5-HT (serotonin) in terminal areas, as measured inmicrodialysis experiments (WO 01/41701). This would imply a shorteronset of antidepressant effect in the clinic and an augmentation orpotentiation of the therapeutic effect of the serotonin reuptakeinhibitor (SRI).

Similarly, it has been reported that the combination of pindolol, whichis a 5-HT_(1A) partial agonist, with a serotonin reuptake inhibitorgives rise to fast onset of effect [Psych. Res., 125, 81-86, 2004].

CNS related diseases, such as e.g. depression, anxiety and schizophreniaare often co-morbid with other disorders or dysfuntionalities, such ascognitive deficits or impairment [Scand. J. Psych., 43, 239-251, 2002;Am. J. Psych., 158, 1722-1725, 2001].

Several neurotransmitters are presumed to be involved in the neuronalevents regulating cognition. In particular, the cholinergic system playsa prominent role in cognition, and compounds affecting the cholinergicsystem are thus potentially useful for the treatment of cognitiveimpairment. Compounds affecting the 5-HT_(1A) receptor and/or the 5-HT₃receptor are known to affect the cholinergic system, and they may assuch be useful in the treatment of cognitive impairment.

Hence, a compound exerting 5-HT_(1A) and/or 5-HT₃ receptor activitywould be expected to be useful in the treatment of cognitive impairment.A compound which moreover also exerts SERT activity would be particularuseful for the treatment of cognitive impairment in depressed patientsas such compound would also provide a fast onset of the treatment of thedepression.

WO 03/029232 discloses e.g. the compound1-[2-(2,4-dimethylphenyl-sulfanyl)phenyl]piperazine (example 1e) as acompound having SERT activity.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine exerts a combinationof SERT inhibition, 5-HT₃ antagonism and 5-HT_(1A) partial agonism.Accordingly, in one embodiment the present invention provides compound Iwhich is 1-[2-(2,4-dimethylphenylsulfanyl)-phenyl]piperazine andpharmaceutically acceptable salts thereof, provided said compound is notthe free base in a non-crystalline form.

In one embodiment, the invention provides the use of compound I intherapy.

In one embodiment, the invention provides a pharmaceutical compositioncomprising compound I.

In one embodiment, the invention provides therapeutic methods comprisingthe administration of an effective amount of compound I to a patient inneed thereof.

In one embodiment, the invention provides the use of compound I in themanufacture of a medicament.

FIGURES

FIG. 1: XRPD of crystalline base

FIG. 2: XRPD of alpha form of hydrobromide salt

FIG. 3: XRPD of beta form of hydrobromide salt

FIG. 4: XRPD of gamma form of hydrobromide salt

FIG. 5: XRPD of hemi hydrate of hydrobromide salt

FIG. 6: XRPD of the mixture of the ethyl acetate solvate and the alphaform of the hydrobromide salt

FIG. 7: XRPD of hydrochloride salt

FIG. 8: XRPD of monohydrate of hydrochloride salt

FIG. 9: XRPD of mesylate salt

FIG. 10: XRPD of fumarate salt

FIG. 11: XRPD of maleate salt

FIG. 12: XRPD of meso-tartrate salt

FIG. 13: XRPD of L-(+)-tartrate salt

FIG. 14: XRPD of D-(−)-tartrate salt

FIG. 15: XRPD of sulphate salt

FIG. 16: XRPD of phosphate salt

FIG. 17: XRPD of nitrate salt

FIGS. 18 a and 18 b: Effect of compounds of the present invention in theintradermal formalin test. X-axis shows the amount of compoundadministered; Y-axis shows the amount of time (sec) spent licking thepaw. FIG. 18 a: Response in the 0-5 minutes period; FIG. 18 b: Responsein the 20-30 minutes period

FIG. 19 a: Extra-cellular acetylcholine levels in prefrontal cortex infreely moving rats upon administration of1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine HBr salt.

FIG. 19 b: Extra-cellular acetylcholine levels in ventral hippocampus infreely moving rats upon administration of1-[2-(2,4-dimethylphenyl-sulfanyl)phenyl]piperazine HBr salt.

FIG. 20: Effect of 1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazineHBr salt on contextual fear conditioning in Sprague-Dawley rats whengiven 60 minutes before acquisition. Freezing behaviour was scoredduring 58-s habituation period prior to the foot shock US (pre-shockacquisition) (white bars). Freezing behaviour was measured 24 h afterthe training (retention test) (black bars).

FIG. 21: Effect of 1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazineHBr salt on contextual fear conditioning in Sprague-Dawley rats whengiven 1 h prior to the retention test. Freezing behaviour was scoredduring 58-s, prior to the foot shock US (acquisition) (white bars).Freezing behaviour was measured 24 h after the training (retention test)(black bars).

FIG. 22: Effect of AA21004 on contextual fear conditioning inSprague-Dawley rats when given immediately after the acquisition.Freezing behaviour was scored during 58-s, prior to the foot shock US(pre-sock acquisition) (white bars). Freezing behaviour was measured 24h after the training (retention test) (black bars).

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to compound I,1-[2-(2,4-dimethylphenylsulfanyl)-phenyl]piperazine, the structure ofwhich is

and pharmaceutically acceptable salts thereof provided compound I is notthe free base in a non-crystalline form.

In one embodiment, said pharmaceutically acceptable salts are acidaddition salts of acids that are non-toxic. Said salts include saltsmade from organic acids, such as maleic, fumaric, benzoic, ascorbic,succinic, oxalic, bis-methylenesalicylic, methanesulfonic,ethanedisulfonic, acetic, propionic, tartaric, salicylic, citric,gluconic, lactic, malic, mandelic, cinnamic, citraconic, aspartic,stearic, palmitic, itaconic, glycolic, p-aminobenzoic, glutamic,benzenesulfonic, theophylline acetic acids, as well as the8-halotheophyllines, for example 8-bromotheophylline. Said salts mayalso be made from inorganic salts, such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric and nitric acids. Particular mentioningis made of salts made from methanesulfonic acid, maleic acid, fumaricacid, meso-tartaric acid, (+)-tartaric acid, (−)-tartaric acid,hydrochloric acid, hydrobromic acid, sulphuric acid, phosphorous acidand nitric acid. Distinct mentioning is made of the hydrobromide salt.

Oral dosage forms, and in particular tablets, are often preferred by thepatients and the medical practitioner due to the ease of administrationand the consequent better compliance. For tablets, it is preferable thatthe active ingredients are crystalline. In one embodiment, the compoundsof the present invention are crystalline.

In one embodiment the crystals of the present invention are solvates,i.e. crystals wherein solvent molecules from part of the crystalstructure. The solvate may be formed from water, in which case thesolvates are often referred to as hydrates. Alternatively, the solvatesmay be formed from other solvents, such as e.g. ethanol, acetone, orethyl acetate. The exact amount of solvate often depends on theconditions. For instance, hydrates will typically loose water as thetemperature is increased or as the relative humidity is decreased.

In one embodiment, the compounds of the present invention are unsolvatedcrystals.

Some compounds are hygroscopic, i.e. the absorb water when exposed tohumidity. Hygroscopicity is generally regarded as an undesired propertyfor compounds that are to be presented in a pharmaceutical formulation,in particular in a dry formulation, such as tablets. In one embodiment,the invention provides crystals with low hygroscopicity. For oral dosageforms using crystalline active ingredients it is also beneficial if saidcrystals are well-defined. In the present context, the term“well-defined” in particular means that the stoichiometry iswell-defined, i.e. that the ratio between the ions forming the salt isthe ratio between small integers, such as 1:1, 1:2, 2:1, 1:1:1, etc. Inone embodiment, the compounds of the present invention are well-definedcrystals.

The crystalline compounds of the present invention may exist in morethan one form, i.e. they may exist in polymorphic forms. Polymorphicforms exist if a compound can crystallize in more than one form. Thepresent invention is intended to encompass all such polymorphic forms,either as pure compounds or as mixtures thereof.

In one embodiment, the compounds of the present invention are in apurified form. The term “purified form” is intended to indicate that thecompound is essentially free of other compounds or other forms of thesame compound, as the case may be.

In one embodiment, the invention provides crystalline salts of compoundsof the present invention with XRPD as shown in FIGS. 1-17, and inparticular FIGS. 2, 3, 4 and 5.

The table below shows the major XRPD reflections for compounds of thepresent invention.

Selected X-ray peak positions (°2θ), All values +−0.1° Crystalline base11.10 16.88 17.42 22.23 hydrobromide (α) 5.85 9.30 17.49 18.58hydrobromide (β) 6.89 9.73 13.78 14.62 hydrobromide (γ) 11.82 16.0117.22 18.84 hydrobromide (hydrate) 10.69 11.66 15.40 17.86 hydrobromide(ethylacetate solvate) 8.29 13.01 13.39 16.62 hydrochloride 9.41 12.3719.66 22.55 hydrochloride (monohydrate) 7.72 13.45 15.39 17.10 mesylate8.93 13.39 15.22 17.09 hydrogenfumarate 5.08 11.32 17.12 18.04hydrogenmaleate 9.72 13.19 14.72 17.88 mesohydrogentartrate 9.51 10.1716.10 25.58 L-(+)-hydrogentartrate 13.32 13.65 14.41 15.80D-(−)-hydrogentartrate 13.32 13.65 14.41 15.80 hydrogen sulphate 11.8217.22 17.72 20.13 dihydrogenphosphate 7.91 11.83 15.69 17.24 nitrate12.50 17.41 18.12 18.47

As evidenced, e.g., by FIGS. 2-5, compounds of the present invention, incasu the hydrobromide salt, may exist in several forms, i.e. bepolymorphic. The polymorphic forms have different properties, and asshown in example 4d. The beta form of the hydrobromide salt is the morestable as demonstrated by the higher DSC melting point and the lowersolubility. Moreover, the beta form has an attractive combination of lowhygroscopicity and solubility, which makes this compound particularlysuited for making tablets. Hence, in one embodiment, the inventionprovides the hydrobromide salt of1-[2-(2,4-dimethylphenylsulphanyl)-phenyl]piperazine with XRPDreflections at approximately 6.89, 9.73, 13.78 and 14.62 (° 2θ), and inparticular with an XRPD as shown in FIG. 3.

The solubility of an active ingredient is also of significance for thechoice of dosage form as it may have a direct impact onbio-availability. For oral dosage forms, a higher solubility of theactive ingredient is generally believed to be beneficial as it increasesthe bio-availability.

Cortical and hippocampal cholinergic neurotransmission are of greatimportance for cognition, and a number of preclinical observations pointto the importance of the serotonin receptor 1A (5-HT_(1A)) for thissystem. T. Koyama in Neurosci. Lett., 265, 33-36, 1999 reports that the5-HT_(1A) agonists BAYX3702 increases the acetylcholin efflux from thecortex and hippocampus in rats. Interestingly, the 5-HT_(1A) antagonistWAY-100635 is capable of eliminating the effect of BAYX3702 showing thatthe effect of BAYX3702 is 5-HT_(1A) mediated.

A number of studies have reported an effect of modulators of 5-HT_(1A)on cognitive impairment. A. Meneses in Neurobiol. Learn. Memory, 71,207-218, 1999 reports that the partial 5-HT₁A agonist(±)-8-hydroxy-2-(di-n-propylamino)-tetralin, HCl (8-OH-DPAT) facilitatesthe consolidation of learning in normal rats and normalises cognitivefunctions in cognitively impaired rats.

These pre-clinical observations seem to be reflected in the clinic, too.T Sumiyoshi in Am. J. Psych., 158, 1722-1725, 2001 reports a studywherein patients received typical anti-psychotics, such as haloperidol,sulpride and pimozide, which all lack 5-HT_(1A) activity in combinationwith placebo or tandospirone, which is a 5-HT_(1A) agonist. Patientsreceiving tandospirone on top of the anti-psychotic showed animprovement in their cognitive performance whereas patients receivingplacebo did not. Similarly, atypical anti-psychotics, such as clozapine,which are also 5-HT_(1A) agonists enhance cognition in schizophrenicpatients, whereas typical anti-psychotics, such as haloperidol whichhave no 5-HT_(1A) activity, do not, Y. Chung, Brain Res., 1023, 54-63,2004.

As mentioned above, the cholinergic system is believed to be involved inthe neuronal events regulating cognition, and the cholinergic system maybe subject to an inhibitory control by the serotonin receptor 3 (5-HT₃)[(Giovannini et al, J Pharmacol Exp Ther 1998, 285:1219-1225; Costalland Naylor, Current Drug Targets—CNS & Neurobiol Disord 2004, 3:27-37)].

In a habituation test in mice, in a T-maze reinforced alternation taskin rats, and in an object discrimination and reversal learning task inthe marmoset, ondansetron reduced the impairment caused by themuscarinic antagonist, scopolamine or lesions of the cholinergicpathways emerging from the nucleus basalis (Barnes et al, PharmacolBiochem Behav 1990, 35: 955-962; Carey et al, Pharamcol Biochem Behav1992, 42: 75-83). Boast et al (Neurobiol Learn Mem 1999, 71: 259-271)used MK-801, a noncompetitive antagonist of the NMDA receptor, todisrupt the cognitive performance of rats trained on a delayednon-matching to sample radial maze task. Ondansetron was shown to blockthe cognitive impairment. Moreover, in a study on the amnesic effect ofethanol in a passive avoidance task in mice, this amnesic effect ofethanol was partially restored to normal by ondansetron(Napiorkowska-Pawlak et al, Fundam Clin pharmacol 2000, 14: 125-131).Thus, facilitation of the cholinergic transmission by 5-HT₃ antagonismafter impairment of the cholinergic system in preclinical models(Diez-Ariza et al, Psychopharmacology 2003, 169: 35-41; Gil-Bea et al,Neuropharmcol 2004, 47: 225-232), suggests a basis for using thistreatment in the therapy of cognitive disorders.

In a randomised double blind crossover study in healthy male subjects,assessments of verbal and spatial memory and sustained attentiondemonstrated that the 5-HT₃ antagonist, alosetron attenuated scopolamineinduced deficits in verbal and spatial memory (Preston, Recent Advancesin the treatment of Neurodegenerative disorders and cognitive function,1994, (eds.) Racagni and Langer, Basel Karger, p. 89-93).

In conclusion, compounds exerting 5-HT_(1A) partial agonistic activityin combination with 5-HT₃ antagonistic activity are believed to beparticular useful for the treatment of cognitive impairment. Compoundswhich moreover exert serotonin reuptake inhibition would be particularuseful for the treatment of cognitive impairment in association withdepression as the serotonin reuptake inhibition in combination with the5-HT_(1A) partial agonism will lead to a faster onset of the effect ofthe treatment of the depression.

As shown in example 1, the compounds of the present invention are potentinhibitors of the human serotonin transporter, i.e. they inhibitserotonin reuptake. Moreover, the compounds are potent antagonists atthe mouse, rat, guinea pig and canine 5-HT₃ receptor. At the human 5-HT₃receptor, cloned into oocytes, the compounds were found to beantagonists at low concentrations (IC₅₀ approx. 30 nM), whilst at higherconcentrations the compounds display agonistic properties (ED₅₀=2.1 μM).A subsequent application of compounds of the present invention at highconcentration did not show any agonistic response, which could be due torapid desenitisation or direct antagonism in vitro. Thus, at lowconcentrations compounds of the present invention display a markedantagonism at the human 5-HT₃ receptor as observed on the 5-HT₃ receptorfrom other species.

Compounds of the present invention bind with very low affinity to the5-HT_(1A) receptor in brain homogenate of both rats and mice. However,the compounds of the present invention bind to the human 5-HT_(1A)receptor with a K_(i) of 40 nM. Moreover, functional data show that thecompounds of the present invention are partial agonists at the human5-HT_(1A) receptor, displaying an efficacy of 85%.

It is anticipated that the activity of the present invention at SERT,5-HT₃-, and 5-HT_(1A) receptors contribute to the in vivo profile of thecompound in humans.

As shown in example 26 the compounds of the present invention give riseto an increase in the extra-cellular level of acetylcholine in theprefrontal cortex and the ventral hippocampus in rats. Thesepre-clinical findings are expected to translate into a clinical effectin the treatment of cognitive impairments, cf. the use of acetylcholineesterase inhibitors in the treatment of cognitive impairments, e.g. inAlzheimer's disease. Further support to this position can be found inexample 27, wherein data show that compounds of the present inventionenhances contextual memory in rats. All in all, the pharmacologicalprofile of the compounds of the present invention combined with theeffects on acetylcholine levels and memory in rats strongly suggest thatthe compounds of the present invention are useful in the treatment ofcognitive impairment.

In one embodiment, the present invention relates to a method for thetreatment of cognitive deficits or cognitive impairment, said methodcomprising the administration of a therapeutically effective amount of acompound of the present invention to a patient in need thereof.

Cognitive deficits or cognitive impairment include a decline incognitive functions or cognitive domains, e.g. working memory, attentionand vigilance, verbal learning and memory, visual learning and memory,reasoning and problem solving e.g. executive function, speed ofprocessing and/or social cognition. In particular, cognitive deficits orcognitive impairment may indicate deficits in attention, disorganizedthinking, slow thinking, difficulty in understanding, poorconcentration, impairment of problem solving, poor memory, difficultiesin expressing thoughts and/or difficulties in integrating thoughts,feelings and behaviour, or difficulties in extinction of irrelevantthoughts. The terms “cognitive deficits” and “cognitive impairment” areintended to indicate the same and are used interchangeably.

In one embodiment, said patient is also diagnosed with another CNSdisorder, such as affective disorders, such as depression; generaliseddepression; major depressive disorder; anxiety disorders includinggeneral anxiety disorder and panic disorder; obsessive compulsivedisorder; schizophrenia; Parkinson's; dementia; AIDS dementia; ADHD; ageassociated memory impairment; or Alzheimer's.

Cognitive impairment is among the classic features of depression, suchas e.g. major depressive disorder. Cognitive disorders may to someextend be secondary to depression in the sense that an improvement inthe depressive state will also lead to an improvement of the cognitiveimpairment. However, there is also clear evidence that cognitivedisorders are, indeed, independent from depression. For instance,studies have shown persistent cognitive impairment upon recovery fromdepression [J. Nervous Mental Disease, 185, 748-754, 197]. Moreover, thedifferential effect of antidepressants on depression and cognitiveimpairments lends further support to the notion that depression andcognitive impairment are independent, albeit often co-morbid conditions.While serotonin and noradrenalin medicaments provide comparableimprovements in depressive symptoms, several studies have shown thatmodulation of the noradrenergic system does not improve the cognitivefunctions as much as serotonin modulation [Brain Res. Bull., 58,345-350, 2002; Hum Psychpharmacol., 8, 41-47, 1993].

The treatment of cognitive impairment in depressed patients by theadministration of the compounds of the present invention is believed tobe particular advantageous. The multifaceted pharmacology of thecompounds of the present invention, in particular the SERT, 5-HT₃ and5-HT_(1A) activity is expected to lead to improvement in cognitivefunctioning in combination with a fast onset treatment of the depressedstate.

Cognitive impairment is a particularly important consideration in theelderly. Cognitive impairment normally increases with age, and furtherwith depression. Hence, in one embodiment, the patient to be treated forcognitive impairment is elderly, and in particular elderly withdepression.

Cognitive functions are, as mentioned above, often impaired inschizophrenic patients. Studies have also concluded that cognitivefunctioning is associated with vocational functioning in schizophrenia[Scizophrenia Res., 45, 175-184, 2000]. In one embodiment, the patientto be treated for cognitive impairment is schizophrenic.

5-HT₃ receptor antagonists have additionally been suggested for thetreatment of diseases such as emesis, chemotherapy-induced emesis,craving, substance abuse, pain, irritable bowel syndrome (IBS),schizophrenia, and eating disorders, [Eur. J. Pharmacol., 560, 1-8,2007; Pharmacol. Therapeut., 111, 855-876, 2006; Alimentary Pharmacol.Ther., 24, 183-205, 2006]

Clinical studies show that a combination of mirtazipine and an SSRI aresuperior to SSRIs alone for the treatment of depressed patients with aninadequate clinical response (treatment resistant depression, TRD, orrefractory depression) [Psychother. Psychosom., 75,139-153, 2006].Mirtazapine is a 5-HT₂ and a 5-HT₃ antagonist, which lends support tothe notion that compounds of the present invention are useful for thetreatment of TRD.

Hot flushes are a symptom associated with the menopausal transition.Some women may suffer from this to an extent where it interferes withsleep or activities in general, and where treatment is necessary.Hormone replacement therapy with oestrogen has been established practicefor decades, however, recently concerns have been voiced on sideeffects, such as breast cancer and cardiac events. Clinical trials withSSRIs have shown that these compounds have an effect on hot flushes,albeit less than for oestrogen [J. Am. Med. Ass., 295, 2057-2071, 2006].Treatment of hot flushes with compounds inhibiting serotonin reuptake,e.g. compounds of the present invention could, however, be analternative treatment for women who can not or will not acceptoestrogen.

Sleep apnoea or obstructive sleep apnoea-hyponea syndrome or obstructivesleep-disordered breathing is a disorder for which an effectivepharmacotherapy remains to be identified. Several studies in animals,however, suggest that 5-HT₃ antagonists, e.g. compounds of the presentinvention may be effective in the treatment of these diseases [Sleep,21, 131-136, 1998; Sleep, 8, 871, 878, 2001].

In one embodiment, the invention relates to a method of treating adisease selected from affective disorders, depression, major depressivedisorder, postnatal depression, depression associated with bipolardisorder, Alzheimer's disease, psychosis, cancer, age or Parkinson'sdisease, anxiety, general anxiety disorder, social anxiety disorder,obsessive compulsive disorder, panic disorder, panic attacks, phobia,social phobia, agoraphobia, stress urinary incontinence, emesis, IBS,eating disorders, chronic pain, partial responders, treatment resistantdepression, Alzheimer's disease, cognitive impairment, ADHD,melancholia, PTSD, hot flushes, sleep apnoea, alcohol, nicotine orcarbohydrate craving, substance abuse and alcohol or drug abuse, themethod comprising the administration of a therapeutically effectiveamount of a compound of the present invention to a patient in needthereof. In one embodiment, said patient being treated for any of theabove listed diseases has initially been diagnosed with said disease.

It is well know that treatment with anti-depressants in general andSSRI's in particular may be associated with sexual dysfunction and whichfrequently leads to discontinuation of the treatment. As much as 30-70%of patients on SSRIs report deficits in sexual function [J. Clin.Psych., 66, 844-848, 2005], which deficits include decreased libido,delayed, reduced or absent orgasms, diminished arousal, and erectiledysfunction. A total of 114 subjects have been exposed to compounds ofthe present invention in clinical trials; of these 114 subjects, onlyone subject reported sexual dysfunction. These data suggest thatclinical intervention using compounds of the present invention isassociated with surprisingly few deficits in sexual functioning.

As mentioned above, compounds of the present invention are particularlywell suited for the treatment of chronic pain. Chronic pain includesindications such as phantom limb pain, neuropathic pain, diabeticneuropathy, post-herpetic neuralgia (PHN), carpal tunnel syndrome (CTS),HIV neuropathy, complex regional pain syndrome (CPRS), trigeminalneuralgia/trigeminus neuralgia/tic douloureux, surgical intervention(e.g. post-operative analgesics), diabetic vasculopathy, capillaryresistance or diabetic symptoms associated with insulitis, painassociated with angina, pain associated with menstruation, painassociated with cancer, dental pain, headache, migraine, tension-typeheadache, trigeminal neuralgia, temporomandibular joint syndrome,myofascial pain muscular injury, fibromyalgia syndrome, bone and jointpain (osteoarthritis), rheumatoid arthritis, rheumatoid arthritis andedema resulting from trauma associated with burns, sprains or fracturebone pain due to osteoarthritis, osteoporosis, bone metastases orunknown reasons, gout, fibrositis, myofascial pain, thoracic outletsyndromes, upper back pain or lower back pain (wherein the back painresults from systematic, regional, or primary spine disease(radiculopathy), pelvic pain, cardiac chest pain, non-cardiac chestpain, spinal cord injury (SCI)-associated pain, central post-strokepain, cancer neuropathy, AIDS pain, sickle cell pain or geriatric pain.

Data presented in example 16 shows that compounds of the presentinvention are useful in the treatment of pain, and that they may evenhave an analgesic effect, additionally studies in an animal model ofneuropathic pain confirm this observation.

A “therapeutically effective amount” of a compound as used herein meansan amount sufficient to cure, alleviate or partially arrest the clinicalmanifestations of a given disease and its complications in a therapeuticintervention comprising the administration of said compound. An amountadequate to accomplish this is defined as “a therapeutically effectiveamount”. Effective amounts for each purpose will depend on the severityof the disease or injury as well as the weight and general state of thesubject. It will be understood that determining an appropriate dosagemay be achieved using routine experimentation, by constructing a matrixof values and testing different points in the matrix, which is allwithin the ordinary skills of a trained physician.

The term “treatment” and “treating” as used herein means the managementand care of a patient for the purpose of combating a condition, such asa disease or a disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the active compound to alleviatethe symptoms or complications, to delay the progression of the disease,disorder or condition, to alleviate or relief the symptoms andcomplications, and/or to cure or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofcombating the disease, condition, or disorder and includes theadministration of the active compounds to prevent the onset of thesymptoms or complications. Nonetheless, prophylactic (preventive) andtherapeutic (curative) treatment are two separate aspects of theinvention. The patient to be treated is preferably a mammal, inparticular a human being.

Typically, the treatment of the present invention will involve dailyadministration of the compounds of the present invention. This mayinvolve once daily administration, or administration twice a day or evenmore frequently.

In one embodiment, the invention relates to the use of a compound of thepresent invention for the manufacture of a medicament for the treatmentof affective disorders, depression, major depressive disorder, postnataldepression, depression associated with bipolar disorder, Alzheimer'sdisease, psychosis, cancer, age or Parkinson's disease, anxiety, generalanxiety disorder, social anxiety disorder, obsessive compulsivedisorder, panic disorder, panic attacks, phobia, social phobia,agoraphobia, stress urinary incontinence, emesis, IBS, eating disorders,chronic pain, partial responders, treatment resistant depression,Alzheimer's disease, cognitive impairment, ADHD, melancholia, PTSD, hotflushes, sleep apnoea, alcohol, nicotine or carbohydrate cravingsubstance abuse, or alcohol or drug abuse.

In one embodiment, the invention relates to compounds of the presentinventions for use in the treatment of a disease selected from affectivedisorders, depression, major depressive disorder, postnatal depression,depression associated with bipolar disorder, Alzheimer's disease,psychosis, cancer, age or Parkinson's disease, anxiety, general anxietydisorder, social anxiety disorder, obsessive compulsive disorder, panicdisorder, panic attacks, phobia, social phobia, agoraphobia, stressurinary incontinence, emesis, IBS, eating disorders, chronic pain,partial responders, treatment resistant depression, Alzheimer's disease,cognitive impairment, ADHD, melancholia, PTSD, hot flushes, sleep apnea,alcohol, nicotine or carbohydrate craving, substance abuse, and alcoholand drug abuse.

The effect of the compounds of the present invention on cognition inhumans may be evaluated in a number of ways. The effect may be evaluatedin tests wherein healthy volunteers are administered the compoundfollowed by a measurement of the cognitive performance in recognisedtests, such as e.g. Auditory Verbal Learning Test (AVLT), Wisconsin CardSorting Test (WCST), or sustained attention, [Psycopharmacol, 163,106-110, 2002; Psychiatry Clin. Neurosci., 60, 70-76, 2006]. The effectmay of course also be assessed in patients suffering from cognitiveimpairment using the same sort of tests. Alternatively, cognitive modelsmay be used wherein cognitive impairment is induced in healthyvolunteers and wherein a restorative effect of the compounds of thepresent invention is measured. Cognitive impairment may be induced bye.g. scopolamine, sleep deprivation, alcohol, and tryptophane depletion.

The pharmaceutical formulations of the invention may be prepared byconventional methods in the art. Particular mentioning is made oftablets, which may be prepared by mixing the active ingredient withordinary adjuvants and/or diluents and subsequently compressing themixture in a conventional tabletting machine. Examples of adjuvants ordiluents comprise: anhydrous calcium hydrogen phosphate, PVP, PVP-VAco-polymers, microcrystalline cellulose, sodium starch glycolate, cornstarch, mannitol, potato starch, talcum, magnesium stearate, gelatine,lactose, gums, and the like. Any other adjuvants or additives usuallyused for such purposes such as colourings, flavourings, preservativesetc. may be used provided that they are compatible with the activeingredients.

Solutions for injections may be prepared by dissolving the activeingredient and possible additives in a part of the solvent forinjection, preferably sterile water, adjusting the solution to desiredvolume, sterilising the solution and filling it in suitable ampules orvials. Any suitable additive conventionally used in the art may beadded, such as tonicity agents, preservatives, antioxidants, etc.

The pharmaceutical compositions of this invention or those which aremanufactured in accordance with this invention may be administered byany suitable route, for example orally in the form of tablets, capsules,powders, syrups, etc., or parenterally in the form of solutions forinjection. For preparing such compositions, methods well known in theart may be used, and any pharmaceutically acceptable carriers, diluents,excipients or other additives normally used in the art may be used.

Conveniently, the compounds of the invention are administered in unitdosage form containing said compounds in an amount of about 1 to 50 mg.An upper limit is believed to be set by the concentration dependency ofthe 5-HT₃ activity. The total daily dose is usually in the range ofabout 1-20 mg, such as about 1 to 10 mg, about 5-10 mg, about 10-20 mg,or about 10-15 mg of the compound of the invention. Particularmentioning is made of daily doses of 5, 10, 15 or 20 mg.

Tablets comprising a compound of the present invention may convenientlybe prepared by wet granulation. Using this method, the dry solids(active ingredients, filler, binder etc.) are blended and moistened withwater or another wetting agent (e.g. an alcohol) and agglomerates orgranules are built up of the moistened solids. Wet massing is continueduntil a desired homogenous particle size has been achieved whereupon thegranulated product is dried. The compounds of the present invention aretypically mixed with lactose monohydrate, corn starch and copovidone ina high shear mixer together with water. Following formation ofgranulates, these granulates may be sieved in a sieve with a suitablesieve size, and dried. The resulting, dried granulates are then mixedwith microcrystalline cellulose, croscarmellose sodium and magnesiumstearate, following which the tablets are pressed. Alternatively, wetgranulation of the compounds of the present invention may be achievedusing mannitol, corn starch and copovidone, which granulates are mixedwith microcrystalline cellulose, sodium starch glycolate and magnesiumstearate before tablets are pressed. Alternatively, wet granulation ofthe compounds of the present invention may be achieved by usinganhydrous calcium hydrogen phosphate, corn starch and copovidone, whichgranulates are mixed with microcrystalline cellulose, sodium starchglycolate (type A), talc and magnesium stearate before tablets arepressed. Copovidone is a PVP-VA copolymer.

In one embodiment, the compound of the present invention is thehydrobromide salt, e.g. in the beta form, and suitable tablets may becomposed as follows—percentages indicated are w/w-%

HBr salt 2-20% Lactose monohydrate 30-50% Starch 15-30% Copovidone 3-5%Microcrystalline cellulose 15-25% Croscarmellose sodium 2-5% Mg stearate0.5-5%

In particular, the tablets may be composed as follows

HBr salt 3-4% Lactose monohydrate 44-46% Starch 22-23% Copovidone 3-4%Microcrystalline cellulose 20-22% Croscarmellose sodium 3-3.5% Mgstearate 0.5-1% or HBr salt 15-16% Lactose monohydrate 35-38% Starch18-20% Copovidone 3-4% Microcrystalline cellulose 20-22% Croscarmellosesodium 3-3.5% Mg stearate 0.5-1% or HBr salt 1-2% Lactose monohydrate44-46% Starch 20-24% Copovidone 3-4% Microcrystalline cellulose 22-24%Croscarmellose sodium 3-4% Mg stearate 0.5-1%

In one embodiment, the compound of the present invention is thehydrobromide salt, e.g. in the beta form, and suitable tablets may becomposed as

HBr salt  2-30% Mannitol 25-45% Corn starch 10-20% Copovidone  2-4%Microcrystalline cellulose 22-27% Sodium starch glycolate  4-5% Mgstearate 0.25-5%, such as 0.25-2%

In particular, the tablets may be composed as follows

HBr salt 20-22% Mannitol 35-36% Corn starch 10-12% Copovidone 2.5-3%Microcrystalline cellulose 24-25% Sodium starch glycolate 3-4% Mgstearate 0.25-1% or HBr salt 12-13% Mannitol 36-37% Corn starch 18-19%Copovidone 3-4% Microcrystalline cellulose 24-25% Sodium starchglycolate 3-4% Mg stearate 0.25-1% or HBr salt 25-27% Mannitol 27-29%Corn starch 13-15% Copovidone 3-4% Microcrystalline cellulose 24-25%Sodium starch glycolate 3-5% Mg stearate 0.25-1% or HBr salt 3-4%Mannitol 40-42% Corn starch 20-22% Copovidone 3-4% Microcrystallinecellulose 26-28% Sodium starch glycolate 3-5% Mg stearate 0.5%In one embodiment, the compound of the present invention is thehydrobromide salt and suitable tablets may be composed as follows

HBr salt 3-8% Anhydrous calcium hydrogen phosphate 35-45% Corn starch15-25% Copovidone 2-6% Microcrystalline cellulose 20-30% Sodium starchglycolate 1-3% Talc 2-6% Magnesium stearate 0.5-2%In particular, the tablets may be composed as follows

HBr salt approximately 5% Anhydrous calcium hydrogen phosphateapproximately 39% Corn starch approximately 20% Copovidone approximately3% Microcrystalline cellulose approximately 25% Sodium starch glycolateapproximately 3% Talc approximately 4% Magnesium stearate approximately1%

Tablets with different amounts of active compound, such as correspondingto e.g., 2.5, 5, 10, 20, 25, 30, 40, 50, 60 or 80 mg of the free basemay be obtained by choosing the right amount of the compound of thepresent invention in combination with a tablet of an appropriate size.

The size of the crystals used for preparing tablets comprising compoundsof the present invention are of significance. If the crystals are toosmall they may stick to the plunger in the tablet machines. On the otherhand, they cannot be too large either. The dissolution rate in theintestines decreases when crystal size increases. Hence, if the crystalsare too large it may compromise the bioavailability of the compounds.Particle size distribution may be described using quantiles, e.g., D5%,D10%, D50%, D90%, D95% and D98%. As used herein, “particle sizedistribution” means the cumulative volume size distribution ofequivalent spherical diameters as determined by laser diffraction at 1bar dispersive pressure in a Sympatec Helos equipment.

In one embodiment, the crystals of the compound of the presentinvention, and in particular the beta from of the hydrobromide salt havea particle size distribution corresponding to D98%: 650-680 μm; D50%:230-250 μm; and D5%: 40-60 μm. In a further embodiment, the particlesize distribution corresponds to D98%: 370-390; d50%: 100-120 μm; D5%:5-15 μm. In a still further embodiment, the particle size distributioncorresponds to D98%: 100-125 μm; D50%: 15-25 μm; and D5%: 1-3 μm. In aneven further embodiment, the particle size distribution corresponds toD98%: 50-70 μm; D50%: 3-7 μm; and D5%: 0.5-2.

The free base of the present invention may be prepared as disclosed inWO 2003/029232. Salts of the present invention may be prepared bydissolving the free base in an appropriate solvent, adding the relevantacid, followed by precipitation. Precipitation may be accomplishedeither by the addition of a second solvent, and/or evaporation, and/orcooling. Alternatively, the free base of the present invention andultimately the compounds of the present invention may be synthesised ina palladium catalysed reaction as described below.

Formation of aromatic carbon-heteroatom bonds may be achieved bynucleophilic aromatic substitution or copper-mediated Ullman reactions.More recently, palladium has been shown to be a powerful catalyst forthe formation of such bonds, and in particular the formation of C—N andC—S bonds, see e.g., U.S. Pat. No. 5,573,460.

In one embodiment, the invention provides a process for the preparationof

or a salt thereof, the process comprising reacting compound II

wherein R′ represents hydrogen or a mono-valent metal ion, with acompound of formula III

wherein X₁ and X₂ independently represent halogen, and a compound offormula IV

wherein R represents hydrogen or a protecting group, in the presence ofa solvent, a base and a palladium catalyst consisting of a palladiumsource and a phosphine ligand at a temperature between 60° C. and 130°C.

In one embodiment, the process is divided in sub-processes whereincompound II and compound III are reacted in a first reaction to providea compound of the formula

This compound is then optionally purified to a suitable degree beforebeing reacted with compound IV to provide4-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine.

One-pot syntheses, i.e. syntheses wherein all reactants are mixedtogether at the start of the reaction or process, are particularlyuseful due to their inherent simplicity. On the other hand, the numberof possible unwanted side-reactions is dramatically increased, whichagain means that the number and/or the amount of unwanted side productsmay increase and the yield of the desired product decreasecorrespondingly. For the present process in particular, it may beobserved that piperazine has two nitrogens each of which couldpotentially participate in the formation of a C—N bond. It hassurprisingly been found that the present process may be run as a one-potsynthesis, i.e. a process wherein compound II, compound III and compoundVI are mixed from the beginning, while maintaining a high yield of apure compound.

Compound II is a thiol or the corresponding thiolate. From aoccupational health perspective is may be beneficial to use a thiolate,such as the Li⁺, Na⁺ or K⁺ thiolate to avoid the odour problemsassociated with thiols. Nonetheless, in one embodiment, R′ is hydrogen.

Compound III is a 1,2-dihalogen activated benzene, and the halogens maybe any of Cl, Br and I. In particular, compound II is1-bromo-2-iodo-benzene or 1,2-dibromo-benzene.

The solvent used in the process of the present invention may be selectedfrom aprotic organic solvents or mixtures of such solvents with aboiling temperature within the reaction temperature range, i.e. 60-130°C. Typically, the solvent is selected from amongst toluene, xylene,triethyl amine, tributyl amine, dioxan, N-methylpyrrolidone, or from anymixture thereof. Particular mentioning is made of toluene as solvent.

Central to the present process is the use of a palladium catalystwithout which the reactions do not take place. The palladium catalystconsists of a palladium source and a phosphine ligand. Useful palladiumsources include palladium in different oxidations states, such as e.g. 0and II. Examples of palladium sources which may be used in the processof the present invention are Pd₂dba₃, Pddba₂ and Pd(OAc)₂. dbaabbreviates dibenzylideneacetone. Particular mentioning is made ofPddba₂ and Pd₂dba₃. The palladium source is typically applied in anamount of 0.1-10 mole-%, such as 1-10 mole-%, such as 1-5 mole-%.Throughout this application, mole-% is calculated with respect to thelimiting reactant.

Numerous phosphine ligands are known, both monedentate and bidentate.Useful phosphine ligands include racemic2,2′-bis-diphenylphosphanyl-[1,1′]binaphtalenyl (rac-BINAP),1,1′-bis(diphenylphosphino)ferrocene (DPPF),bis-(2-diphenylphosphinophenyl)ether (DPEphos), tri-t-butyl phosphine(Fu's salt), biphenyl-2-yl-di-t-butyl-phosphine,biphenyl-2-yl-dicyclohexyl-phosphine,(2′-dicyclohexylphosphanyl-biphenyl-2-yl)-dimethyl-amine,[2′-(di-t-butyl-phosphanyl)-biphenyl-2-yl]-dimethyl-amine, anddicyclohexyl-(2′,4′,6′-tri-propyl-biphenyl-2-yl)-phosphane. Moreover,carbene ligands, such as e.g.1,3-bis-(2,6-di-isopropyl-phenyl)-3H-imidazol-1-ium; chloride may beused in stead of phosphine ligands. In one embodiment, the phosphineligand is rac-BINAP, DPPF or DPEphos, and in particular rac-BINAP. Thephosphine ligand is usually applied in an amount between 0.1 and 10mole-%, such as 1 and 5 mole-%, typically around 1-2 mole-%.

Base is added to the reaction mixture to increase pH. In particularbases selected from NaOt-Bu, KOt-Bu and Cs₂CO₃ are useful. Organicbases, such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and1,4-diazabicyclo[2.2.2]octane (DABCO) may be applied as well. Particularmentioning is made of NaO(t-Bu) and KO(t-Bu). Typically, the base isadded is an amount around 1-5 equivalents, such as 1-3 equivalents, suchas 2-3 equivalents.

Compound IV is a piperazine compound. Piperazine has two nitrogens, onlyone of which is to participate in the C—N bond formation. In oneembodiment, formation of bonds to the second nitrogen is avoided byusing a mono-protected piperazine, i.e. an embodiment wherein R is aprotective group. Many protective groups are known in the art, anduseful examples include boc, Bn, Cbz, C(═O)O and Me, and in particularboc. Bn abbreviates benzyl; boc abbreviates t-butyloxycarbonyl; and cbzabbreviates benzyloxycarbonyl. If a protected piperazine is used in thereactions, the protecting group has to be removed in a subsequent step,typically by the addition of aqueous acid. If methyl is used as theprotecting group, the methyl may be removed in a reaction with carbamateand subsequent removal of this group.

It has surprisingly been found that unprotected piperazine may be usedas well without the formation of unwanted bonds to the second nitrogen.Protected and unprotected piperazine have different solubilities indifferent solvents; as an example, piperazine is practically insolublein toluene whereas boc protected piperazine is highly soluble intoluene. Normally it would be expected that it is a requirement for asuccessful reaction that all reactants are readily soluble in theapplied solvent. Nevertheless, it has been found that the process of thepresent invention runs with a high yield with toluene as solvent andwith unprotected piperazine, i.e. in an embodiment wherein R ishydrogen. Hence, in one embodiment, the solvent is toluene and compoundIV is piperazine. In a further embodiment, this combination ofconditions is used in a one-pot synthesis.

In one embodiment, the temperature under which to run to process isapproximately 80° C.-approximately 120° C.

In one embodiment, 1-[2-(2,4-dimethyl-phenylsulfanyl)phenyl]-piperazineis prepared in a process comprising the following steps

a. Dissolving or dispersing 1-1.5 equivalents of compounds II, III andIV in toluene to obtain mixture A;

b. Adding 1-2 mole-% of Pddba₂ and 1-2 mole-% of rac-BINAP together with2-3 equivalents of NaOt-Bu, optionally dispersed or dissolved ordispersed in toluene, to mixture A to obtain mixture B, which is heatedto around 100° C. until compound II and III are fully converted,typically 5-10 hours;

c. Increasing the temperature of the mixture obtained in step b toaround 120° C. until compound IV is fully converted, typically 16-32hours; and

d. Optionally removing the protecting group by the addition of aqueousacid if compound III is a protected piperazine.

Optionally, purifications steps may be included in the above sequence ofreaction steps.

In one embodiment, 1-1.5 equivalents of 2,4-dimethyl-thiol,1-bromo-2-iodo benzene (or 1,2-dibromo-benzene) and piperazine isdispersed in toluene followed by the addition of 2-5, such as 3equivalents NaOt-Bu and 1-2 Mole-% Pd₂dba₃ and rac-BINAP dispersed intoluene to obtain a mixture which is refluxed for 2-10 hours, typically3-5 hours to obtain1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine. Optionally, thisproduct may be further reacted with aqueous HBr to achieve thecorresponding hydrobromic acid addition salt.

In one embodiment, 2-5 equivalents of NaOt-Bu, 2-5 equivalentspiperazine, 0.2-0.6 mole-% Pddba₂, and 0.6-1 mole-% rac-BINAP isdispersed in toluene to obtain mixture A′, to which mixtureapproximately 1 equivalent 2-bromo-iodobezene is added to obtain mixtureB′, to which mixture 1 equivalent 2,4-dimethylthiophenol is added andthe resulting mixture is heated to reflux for 3-7 hours, such as 4-6hours to obtain 1-[2-(2,4-dimethyl-phenylsulfanyl)-phenyl]-piperazine.Optionally, this product may be further reacted with aqueous HBr toachieve the corresponding hydrobromic acid addition salt.

In some situations, it may be desirable to obtain an acid addition saltof 1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine rather than thefree base. Acid addition salts may be achieved in a further process stepin which the free base obtained is reacted with a relevant acid, such ase.g. fumaric acid, sulphuric acid, hydrochloric acid or hydrobromicacid. The acid may be added directly to the reaction mixture or,alternatively, the free base may be purified to any suitable degreeinitially before such step. If the free base has been isolated as a drycompound, it may be necessary to use a solvent in order to bring thefree base into solution prior to a reaction with the acid. In oneembodiment, aqueous hydrobromic acid is added directly to the reactionmixture without any initial purification of the free base.

In processes wherein a protected piperazine has been used, theprotecting group has to be removed by the addition of an aqueous acid asexplained above. In one embodiment, said aqueous acid may be selected toachieve two transformations, i.e. the de-protection of the protectedpiperazine and the formation of an acid addition salt. In particular,aqueous hydrobromic acid may be used to de-protect protected piperazineand to obtain the hydrobromic acid addition salt in one process step.

It goes for all the reactions and reaction mixtures mentioned here thatit may be an advantage to purge them with an inert gas or run them undera blanket of inert gas. Nitrogen is a cheap and readily availableexample of an inert gas

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. For example, the phrase “the compound”is to be understood as referring to various compounds of the inventionor particular described aspect, unless otherwise indicated.

Unless otherwise indicated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The description herein of any aspect or aspect of the invention usingterms such as “comprising”, “having,” “including,” or “containing” withreference to an element or elements is intended to provide support for asimilar aspect or aspect of the invention that “consists of”, “consistsessentially of”, or “substantially comprises” that particular element orelements, unless otherwise stated or clearly contradicted by context(e.g., a composition described herein as comprising a particular elementshould be understood as also describing a composition consisting of thatelement, unless otherwise stated or clearly contradicted by context).

EXAMPLES Analytical Methods

¹H NMR spectra are recorded at 500.13 MHz on a Bruker Avance DRX500instrument. Dimethyl sulfoxide (99.8% D) is used as solvent, andtetramethylsilane (TMS) is used as internal reference standard.

The melting points are measured using Differential Scanning calorimetry(DSC). The equipment is a TA-Instruments DSC-Q1000 calibrated at 5°/minto give the melting point as onset value. About 2 mg of sample is heated5°/min in a loosely closed pan under nitrogen flow.

Thermo gravimetric analysis (TGA) used for estimation of solvent/watercontent of dried material is performed using a TA-instruments TGA-Q500.1-10 mg sample is heated 10°/min in an open pan under nitrogen flow.

X-Ray powder diffractograms were measured on a PANalytical X'Pert PROX-Ray Diffractometer using CuK_(α1) radiation. The samples were measuredin reflection mode in the 2θ-range 5-40° using an X'celerator detector.

Example 1 In Vitro Receptor Pharmacology

Rat serotonin transporter: IC₅₀ 5.3 nM (blockade of 5-HT uptake)

Human serotonin transporter: IC₅₀ 40 nM (blockade of 5-HT uptake)

Human 5-HT_(1A) receptor: K_(i) 40 nM with partial agonism (efficacy85%)

Rat 5-HT₃ receptor: IC₅₀ 0.2 nM (antagonism in functional assay)

Human 5-HT_(3A) receptor: IC₅₀ around 20 nM (antagonism in functionalassay). At higher concentration, the compound exhibits agonisticactivity with an ED₅₀ of 2.1 μM. The compound of the invention alsoshowed high affinity for the human 5HT₃ receptor in an in vitro bindingassay (Ki 4.5 nM).

Example 2 Cognitive Effects

As discussed above, the compounds of the present invention interact withthe cholinergic system, and it would be expected to see an effect in oneor more of the following in vivo models.

-   -   Five choice serial reaction time test (5-CSRT), which is useful        for demonstrating an effect on continuous attention    -   Spatial Y maze test, which is useful for demonstrating effects        on short, long-term and working memory    -   Attentional set shifting model, which is useful for        demonstrating effects on executive functioning, i.e. reasoning        and problem solving

Example 3a Preparation of the Free Base of Compound I

10 grams of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazinehydrobromide was treated with a stirred mixture of 100 ml 3 M NaOH and100 ml ethyl acetate for 10 minutes. The organic phase was separated,washed with 100 ml 15%-wt NaCl (aq), dried over MgSO₄, filtered andconcentrated in vacuum producing 7.7 gram (98%) of compound I base as aclear colourless oil.

NMR complies with structure.

Example 3b Preparation of Crystalline Base of Compound I

3.0 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazinecolourless oil was treated with 70 ml acetonitrile and heated to reflux.The almost clear solution was filtered and the clear filtrate was cooledspontaneously upon which precipitation began shortly after filtration.The mixture was stirred at room temperature (22° C.) for 2 hours and theproduct was isolated by filtration and dried in vacuum (40° C.)overnight. The crystalline base was isolated as a white solid in 2.7gram (90%). NMR complies with structure. Elemental analysis: 72.40% C,9.28% N, 7.58% H (theory: 72.26% C, 9.36% N, 7.42% H)

Example 3c Characterisation of Crystalline Base of Compound I

The base, as prepared in example 3b, is crystalline (XRPD)—see FIG. 1.It has a melting point of ˜117° C. It is not hygroscopic and has asolubility of 0.1 mg/ml in water.

Example 4a Preparation of the Alpha Form of the Hydrobromide Salt ofCompound I

2.0 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine wasdissolved in hot 30 ml ethyl acetate and added 0.73 ml 48%-wt HBr (aq).This addition caused formation of a thick slurry and additional 10 mlethyl acetate was added in order to have proper stirring. The slurry wasstirred at room temperature for one hour. Filtration and drying invacuum (20° C.) over night produced 2.0 gram of the product as a whitesolid (80%). NMR complies with structure. Elemental analysis: 57.05% C,7.18% N, 6.16% H (Theory for 1:1 salt: 56.99% C, 7.39% N, 6.11% H)

Example 4b Characterisation of the Alpha Form of the Hydrobromide ofCompound I

The alpha form of the hydrobromide, as prepared in example 4a, iscrystalline (XRPD)—see FIG. 2. It has a melting point of ˜226° C. Itabsorbs about 0.3% of water when exposed to high relative humidity andhas a solubility of 2 mg/ml in water.

Example 4c Preparation of the Beta Form of the Hydrobromide Salt ofCompound I

49.5 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazinecolourless oil was dissolved in 500 ml ethyl acetate and added 18.5 ml48%-wt HBr (aq). This addition caused formation of a thick slurry whichwas stirred over night at room temperature. Filtration and drying invacuum (50° C.) over night produced the product in 29.6 gram as whitesolid (47%).

NMR complies with structure. Elemental analysis: 56.86% C, 7.35% N,6.24% H (Theory for 1:1 salt: 56.99% C, 7.39% N, 6.11% H)

Example 4d Characterisation of the Beta Form of the Hydrobromide ofCompound I

The beta form of the hydrobromide, as prepared in example 4c, iscrystalline (XRPD) see FIG. 3. It has a melting point of ˜231° C. Itabsorbs about 0.6% of water when exposed to high relative humidity andhas a solubility of 1.2 mg/ml in water.

Example 4e Preparation of the Gamma Form of the Hydrobromide Salt ofCompound I

1 g of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine hydrobromideas prepared in example 4a was added 20 ml water and heated to 85° C. Thesolution was almost clear. Addition of 1 drop of HBr made it clear. HBrwas added until cloud point was observed. The solution was cooled toroom temperature and dried. NMR complies with structure. Elementalanalysis: 56.63% C, 7.18% N, 6.21% H (Theory for 1:1 salt: 56.99% C,7.39% N, 6.11% H)

Example 4f Characterisation of the Gamma Form of the Hydrobromide ofCompound I

The hydrobromide, as prepared in example 6e, is crystalline (XRPD)—seeFIG. 4. The DSC curve shows some thermal events at about 100° C.;probably change in crystal form. Then it melts at about 220° C. Itabsorbs about 4.5% of water when exposed to high relative humidity andat 30% RH at room temperature about 2% of water is absorbed.

Example 4g Preparation of the Hydrobromide Hydrate of Compound I

1.4 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil wasadded 20 ml water, and heated to 60° C. pH was adjusted to 1 using 48%HBr. The solution was cooled to room temperature and dried. NMR complieswith structure. Elemental analysis: 55.21% C, 7.16% N, 6.34% H (Theoryfor 1:1 salt hemihydrate: 55.68% C, 7.21% N, 6.23% H)

Example 4h Characterisation of the Hemi Hydrate of the Hydrobromide ofCompound I

The hydrate as prepared in Example 4g is crystalline (XRPD)—see FIG. 5.The water content depends strongly on the relative humidity. At roomtemperature and 95% RH the water content is about 3.7%. Dehydrationoccurs by heating to about 100° C.

Example 4i Preparation of the Ethyl Acetate Solvate of the HydrobromideSalt of Compound I

0.9 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil wasdissolved in 35 ml ethyl acetate and added 0.5 ml 48%-wt HBr (aq). Thisaddition caused formation of a thick slurry which was stirred over nightat room temperature. Filtration and washing with 30 ml diethyl etherfollowed by drying in vacuum (50° C.) over night produced1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine HBr EtOAc solvate in1.0 gram (65%). NMR complies with structure. Elemental analysis: 56.19%C, 6.60% N, 6.56% H (Theory for 1:1 salt when corrected for 8% of Ethylacetate and 0.5% water as determined by TGA and KF: 56.51% C, 6.76% N,6.38% H)

Example 4j Characterisation of the Ethyl Acetate Solvate of theHydrobromide of Compound I

The ethyl acetate solvate, as prepared in example 4i, is crystalline(XRPD)—see FIG. 6. The batch contains a mixture of the solvate and thealpha form of compound I, probably because the drying has caused partlydesolvation. The desolvation starts at ˜75° C. when heated 10°/min.After desolvation the alpha form is formed.

If exposed to high relative humidity, the ethyl acetate is replaced bywater, which is released when the humidity subsequently is lowered. Theresulting solid is hygroscopic and absorbs 3.2% of water at highrelative humidity.

Example 5a Preparation of Hydrochloride Salt of Compound I

1.0 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil wasdissolved in 20 ml ethyl acetate using gentle heating (30° C.). When aclear solution was obtained a solution of 2 M HCl in diethyl ether wasadded slowly until pH was approximately 1-2. During the additionspontaneous precipitation was observed. After final addition thesuspension was stirred for 1 hour before the white precipitate wasisolated by filtration and dried in vacuum (40° C.) overnight.1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine hydrochloride wasisolated in 1.1 gram (99%).

NMR complies with structure. Elemental analysis: 64.18% C, 8.25% N,6.96% H (Theory for 1:1 salt when corrected for 0.66% of water asdetermined by TGA: 64.13% C, 8.31% N, 6.95% H)

Example 5b Characterisation of the Hydrochloride of Compound I

The hydrochloride, as prepared in example 5a, is crystalline (XRPD)—seeFIG. 7. It has a melting point of ˜236° C. It absorbs about 1.5% ofwater when exposed to high relative humidity and has a solubility of 3mg/ml in water.

Example 5c Preparation of the Hydrochloride Monohydrate of Compound I

11.9 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil wasdissolved in 100 ml ethanol using heating. When a homogenous solutionwas obtained addition of 3.5 ml conc. HCl (aq) took place causing theimmediately precipitation of a white solid. The suspension was stirredfor 5 minutes at first and then on ice-bath another hour beforefiltration. The white solid was washed using 100 ml of fresh coolethanol (placed in freezer at −18° C. for 2 hours), 50 ml acetone andfinally 50 ml diethyl ether before dried in vacuum (50° C.) overnight.1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine HCl was isolated in5.1 gram (38%). NMR complies with structure. Elemental analysis: 61.23%C, 7.91% N, 7.16% H (Theory for 1:1 salt monohydrate: 61.26% C, 7.94% N,7.14% H)

Example 5d Characterisation of the Hydrochloride Monohydrate of CompoundI

The hydrochloride monohydrate, as prepared in example 5c, is crystalline(XRPD)—see FIG. 8. It dehydrates starting at about 50° C. Some thermalevents, probably rearrangement, occur by further heating, and it meltsat about 230° C. followed by recrystallisation and melting at about 236°C. It does not absorb further amount of water when exposed to highrelative humidity and the hydrate bounded water is not released untilthe relative humidity is decreased to below 10% RH at room temperature.It has a solubility of about 2 mg/ml in water.

Example 6a Preparation of Mesylate Salt of Compound I

1.0 gram of 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil wasdissolved in 20 ml ethyl acetate by heating (70° C.). When a clearsolution was obtained 0.35 gram of methane sulphonic acid (1.1 eqv.) wasadded slowly. After final addition the solution was cooled on ice anddiethyl ether was added slowly causing the precipitation of the product.The suspension was stirred for 2 hours on ice before the whiteprecipitate was isolated by filtration and dried in vacuum (40° C.)overnight. 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine mesylatewas isolated in 1.1 gram (85%). NMR complies with structure. Elementalanalysis: 57.81% C, 6.81% N, 6.68% H (Theory for a 1:1 salt: 57.81% C,7.10% N, 6.64% H)

Example 6b Characterisation of the Mesylate of Compound I

The mesylate, as prepared in example 7a, is crystalline (XRPD)—see FIG.9. It has a melting point of ˜163° C. It is hygroscopic (absorbs about8% of water when exposed to 80% relative humidity and is therebytransformed into a hydrated form. The last 6% of the absorbed water isnot released until the relative humidity is below 10% RH. It has a veryhigh solubility in water (>45 mg/ml).

Example 7a Preparation of Fumarate of Compound I

5.5 gram 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil washeated to reflux in a mixture of 50 ml methanol and 50 ml ethyl acetate.The solution was left to cool slightly before addition of 2.1 gramfumaric acid took place causing an exothermic reaction and precipitationof a white solid. The suspension was stirred while being allowed to coolto room temperature followed by 2 hours in the freezer at −18° C. Thewhite solid was collected by filtration and washed with 20 ml cold ethylacetate before drying in vacuum (50° C.) over night. The product wasisolated in 3.1 gram (44%).

NMR complies with structure. Elemental analysis: 63.42% C, 6.64% N,6.42% H (Theory for a 1:1 salt: 63.74% C, 6.76% N, 6.32% H)

Example 7b Characterisation of the Fumarate of Compound I

The fumarate, as prepared in example 7a, is crystalline (XRPD)—see FIG.10. It has a melting point of ˜194° C. The solubility in water is 0.4mg/ml.

Example 8a Preparation of Maleate of Compound I

2.5 gram 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine oil wasdissolved in 50 ml ethyl acetate and heated to 60° C. followed byaddition of 1.1 gram maleic acid. The mixture was heated again to refluxfor 5 minutes and left to cool to room temperature while stirring.During the cooling precipitation started and was finalized by 4 hours inthe freezer (−18° C.). The white solid was collected by filtration andwashed with 50 ml diethyl ether before drying in vacuum (50° C.) overnight. This produced 1.3 gram of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine Maleate (38%) thatwas recrystallised by treatment with 40 ml ethyl acetate and 5 mlmethanol at reflux. The clear solution was cooled to room temperaturefollowed by 2 hours in the freezer (−18° C.) before filtering and washedtwice with 10 ml cold ethyl acetate followed by drying in vacuum (50°C.) for two days. 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazineMaleate was isolated in 0.9 gram (69%).

NMR complies with structure. Elemental analysis: 63.57% C, 6.79% N,6.39% H (Theory for a 1:1 salt: 63.74% C, 6.76% N, 6.32% H)

Example 8b Characterisation of the Maleate of Compound I

The maleate, as prepared in example 8a, is crystalline (XRPD)—see FIG.11. It has a melting point of ˜152° C. The solubility in water is ˜1mg/ml.

Example 9a Preparation of Meso-Tartrate of Compound I

11.1 ml of a 0.30 M solution of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine in acetone wastreated with 0.5 gram meso-tartaric acid dissolved in 5 ml acetone. Themixture was stirred at room temperature for 30 minutes during whichprecipitation took place. Filtration and washing first with 5 ml acetoneand then 3 ml diethyl ether produced the product as a white solid thatwas dried in vacuum (50° C.) over night.1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine meso-tartaric acidwas isolated in 1.4 gram (93%). NMR complies with structure. Elementalanalysis: 58.58% C, 6.29% N, 6.40% H (Theory for a 1:1 salt: 58.91% C,6.25% N, 6.29% H)

Example 9b Characterisation of the Meso-Tartrate of Compound I

The meso-tartrate, as prepared in example 9a, is crystalline (XRPD)—seeFIG. 12. It has a melting point of ˜164° C. The solubility in water is˜0.7 mg/ml.

Example 10a Preparation of L-(+)-Tartrate of Compound I

11.1 ml of a 0.30 M solution of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine in acetone wastreated with 0.5 gram L-(+)-tartaric acid dissolved in 5 ml acetone. Themixture was stirred at room temperature for 30 minutes during whichprecipitation took place. Filtration and washing first with 5 ml acetoneand then 3 ml diethyl ether achieved the product as a white solid thatwas dried in vacuum (50° C.) over night.1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine (+)-tartaric acidwas isolated in 1.2 gram (81%). NMR complies with structure. Elementalanalysis: 58.86% C, 6.30% N, 6.38% H (Theory for a 1:1 salt: 58.91% C,6.25% N, 6.29% H)

Example 10b Characterisation of the L-(+)-Tartrate of Compound I

The L-(+)-tartrate, as prepared in example 10a, is crystalline(XRPD)—see FIG. 13. It has a melting point of ˜171° C. The solubility inwater is ˜0.4 mg/ml.

Example 11a Preparation of D-(−)-Tartrate of Compound I

11.1 ml of a 0.30 M solution of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine in acetone wastreated with 0.5 gram D-(−)-tartaric acid dissolved in 5 ml acetone. Themixture was stirred at room temperature for 30 minutes during whichprecipitation took place. Filtration and washing first with 5 ml acetoneand then 3 ml diethyl ether produced the product as a white solid thatwas dried in vacuum (50° C.) over night.1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine D-(−)-tartaric acidwas isolated in 1.0 gram (68%). NMR complies with structure. Elementalanalysis: 58.90% C, 6.26% N, 6.35% H (Theory for a 1:1 salt: 58.91% C,6.25% N, 6.29% H)

Example 11b Characterisation of the D-(−)-Tartrate of Compound I

The D-(+)-tartrate, as prepared in example 11a, is crystalline(XRPD)—see FIG. 14. It has a melting point of ˜175° C. The solubility inwater is ˜0.4 mg/ml.

Example 12a Preparation of Sulphate of Compound I

11.1 ml of a 0.30 M solution of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine in acetone wastreated with 2.2 ml of a 3 M solution of H₂SO₄ (aq). The mixture wasstirred at room temperature for 30 minutes and then on ice-bath foranother 4 hours before precipitation took place and was finalized.Filtration and washing first with 5 ml acetone and then 3 ml diethylether produced the product as a white solid that was dried in vacuum(50° C.) over night. 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazinesulphate was isolated in 0.51 gram (39%). NMR complies with structure.Elemental analysis: 54.53% C, 7.22% N, 6.28% H (Theory for a 1:1 salt:54.52% C, 7.07% N, 6.10% H)

Example 12b Characterisation of the Sulphate of Compound I

The sulphate, as prepared in example 12a, is crystalline (XRPD)—see FIG.15. It has a melting point of ˜166° C. The solubility in water is ˜0.1mg/ml.

Example 13a Preparation of Phosphate of Compound I

11.1 ml of a 0.30 M solution of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine in acetone wastreated with 0.2 ml 65% H₃PO₄ (aq). The mixture was stirred at roomtemperature for 30 minutes during which precipitation took place.Filtration and washing first with 5 ml acetone and then 3 ml diethylether produced the product as a white solid that was dried in vacuum(50° C.) over night. 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazinephosphate was isolated in 1.23 gram (94%). NMR complies with structure.Elemental analysis: 54.21% C, 7.15% N, 6.43% H (Theory for a 1:1 salt:54.53% C, 7.07% N, 6.36% H)

Example 13b Characterisation of the Phosphate of Compound I

The phosphate, as prepared in example 13a, is crystalline (XRPD) seeFIG. 16. It has a melting point of ˜224° C. The solubility in water is˜1 mg/ml.

Example 14a Preparation of Nitrate of Compound I

11.1 ml of a 0.30 M solution of1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazine in acetone wastreated with 0.2 ml of 16.5 M HNO₃ (aq). The mixture was stirred at roomtemperature for 30 minutes during which precipitation took place.Filtration and washing first with 5 ml acetone and then 3 ml diethylether produced the product as a white solid that was dried in vacuum(50° C.) over night. 1-[2-(2,4-Dimethylphenylsulfanyl)-phenyl]piperazinenitrate was isolated in 0.87 gram (73%). NMR complies with structure.Elemental analysis: 59.80% C, 11.67% N, 6.51% H (Theory for a 1:1 salt:59.81% C, 11.63% N, 6.41% H)

Example 14b Characterisation of the Nitrate of Compound I

The nitrate, as prepared in example 14a, is crystalline (XRPD)—see FIG.17. It does not melt but decomposes under an exothermic reaction atabout 160° C. The solubility in water is ˜0.8 mg/ml.

Example 15 Tablet

The examples below show representative examples of how tabletscomprising compounds of the present invention may be prepared. The betaform of the hydrobromide salt has been used in all examples.

Example 15a

63.55 g of the hydrobromide salt, 923.65 g Lactosum 350M, 461.8 g cornstarch and 76.0 g Kollidon VA64 were mixed for 2 minutes in a Diosna PP1high shear mixer at an impeller speed of 1000 rpm. Next, the speed ofthe impeller was lowered to 800 rpm and 220 g water was added during thecourse of a minute. Massing was performed for 7 minutes and theresulting granules were passed through a sieve, size 4000 μm. Thegranules were dried and passed through a sieve, size 710 μm. 1383.5 g ofthe resulting granules were mixed with 400 g Avicel PH200 and 60 gAc-Di-Sol. Following lubrication of the blend by mixing with 15 gmagnesium stearate the powder blend was transferred to a tablet press.Tablets having a target core weight of 200 mg and a diameter of 8 mmwere prepared to obtain tablets with a target content corresponding to 5mg of the free base.

Example 15b

317.75 g of the hydrobromide salt, 754.15 g Lactosum 350M, 377.1 g cornstarch and 76.0 g Kollidon VA64 were mixed for 2 minutes in a Diosna PP1high shear mixer at an impeller speed of 1000 rpm. Next, the speed ofthe impeller was lowered to 800 rpm and 210 g water was added during thecourse of a minute. Massing was performed for 7 minutes and theresulting granules were passed through a sieve, size 4000 μm. Thegranules were dried and passed through a sieve, size 710 μm. 1386.2 g ofthe resulting granules were mixed with 400 g Avicel PH200 and 60 gAc-Di-Sol. Following lubrication of the blend by mixing with 15 gmagnesium stearate the powder blend was transferred to a tablet press.Tablets having a target core weight of 200 mg and a diameter of 8 mmwere prepared to obtain tablets with a target content corresponding to25 mg of the free base.

Example 15c

32.2 g of the hydrobromide salt, 944.82 g Lactosum 350M, 472.4 g cornstarch and 76.0 g Kollidon VA64 were mixed for 2 minutes in a Diosna PP1high shear mixer at an impeller speed of 1000 rpm. Next, the speed ofthe impeller was lowered to 800 rpm and 220 g water was added during thecourse of a minute. Massing was performed for 7 minutes and theresulting granules were passed through a sieve, size 4000 μm. Thegranules were dried and passed through a sieve, size 710 μm. 1317 g ofthe resulting granules were mixed with 400 g Avicel PH200 and 60 gAc-Di-Sol. Following lubrication of the blend by mixing with 15 gmagnesium stearate the powder blend was transferred to a tablet press.Tablets having a target core weight of 208 mg and a diameter of 8 mmwere prepared to obtain tablets with a target content corresponding to2.5 mg of the free base.

Example 15d

540.85 g of the hydrobromide salt, 953.00 g Pearlitol 50C, 296.22 g cornstarch and 70.5 g Kollidon VA64 were mixed for 2 minutes in anAeromatic-Fielder PMA1 high shear mixer at an impeller speed of 1000rpm. Next, the speed of the impeller was lowered to 800 rpm and 241.87 gwater was added during the course of a minute. Massing was performed for7 minutes and the resulting granules were passed through a sieve, size4000 μm. The granules were dried and passed through a sieve, size 710μm. 1500 g of the resulting granules were mixed with 531.91 g AvicelPH200 and 85.11 g Primojel. Following lubrication of the blend by mixingwith 10.64 g magnesium stearate the powder blend was transferred to atablet press. Tablets having a target core weight of 125 mg and adiameter of 6 mm were prepared to obtain tablets with a target contentcorresponding to 25 mg of the free base.

Example 15e

270.45 g of the hydrobromide salt, 772.0 g Pearlitol 50C, 386.41 g cornstarch and 70.5 g Kollidon VA64 were mixed for 2 minutes in anAeromatic-Fielder PMA1 high shear mixer at an impeller speed of 1000rpm. Next, the speed of the impeller was lowered to 800 rpm and 195 gwater was added during the course of a minute. Massing was performed for5.5 minutes and the resulting granules were passed through a sieve, size4000 μm. The granules were dried and passed through a sieve, size 710μm. 1200.3 g of the resulting granules were mixed with 425.5 g AvicelPH200 and 68.09 g Primojel. Following lubrication of the blend by mixingwith 8.8 g magnesium stearate the powder blend was transferred to atablet press. Tablets having a target core weight of 100 and a diameterof 6 mm were prepared to obtain tablets with a target contentcorresponding to 10 mg of the free base.

Example 15f

504.85 g of the free base, 552.95 g Pearlitol 50C, 276.53 g corn starchand 65.7 g Kollidon VA64 were mixed for 2 minutes in anAeromatic-Fielder PMA1 high shear mixer at an impeller speed of 1000rpm. Next, the speed of the impeller was lowered to 800 rpm and 182 gwater was added during the course of a minute. Massing was performed for5.5 minutes and the resulting granules were passed through a sieve, size4000 μm. The granules were dried and passed through a sieve, size 710μm. 1250.7 g of the resulting granules were mixed with 443.31 g AvicelPH200 and 70.8 g Primojel. Following lubrication of the blend by mixingwith 8.92 g magnesium stearate the powder blend was transferred to atablet press. Tablets having a target core weight of 250 mg and adiameter of 8 mm were prepared to obtain tablets with a target contentcorresponding to 50 mg of the free base.

Example 15g

135.23 g of the hydrobromide salt, 863.2 g Pearlitol 50C, 432.69 g cornstarch and 70.66 g Kollidon VA64 were mixed for 2 minutes in anAeromatic-Fielder PMA1 high shear mixer at an impeller speed of 1000rpm. Next, the speed of the impeller was lowered to 800 rpm and 195 gwater was added during the course of a minute. Massing was performed for5.5 minutes and the resulting granules were passed through a sieve, size4000 μm. The granules were dried and passed through a sieve, size 710μm. 1200 g of the resulting granules were mixed with 425.28 g AvicelPH200 and 68.2 g Primojel. Following lubrication of the blend by mixingwith 8.58 g magnesium stearate the powder blend was transferred to atablet press. Tablets having a target core weight of 100 mg and adiameter of 6 mm were prepared to obtain tablets with a target contentof corresponding to 5 mg of the free base.

Example 15h

67.6 g of the hydrobromide salt, 908.0 g Pearlitol 50C, 453.9 g cornstarch and 70.51 g Kollidon VA64 were mixed for 2 minutes in a Diosna PP1 high shear mixer at an impeller speed of 1000 rpm. Next, the speed ofthe impeller was lowered to 800 rpm and 195 g water was added during thecourse of a minute. Massing was performed for 5.5 minutes and theresulting granules were passed through a sieve, size 4000 μm. Thegranules were dried and passed through a sieve, size 710 μm. 1325 g ofthe resulting granules were mixed with 531.91 g Avicel PH200 and 85.11 gPrimojel. Following lubrication of the blend by mixing with 10.64 gmagnesium stearate the powder blend was transferred to a tablet press.Tablets having a target core weight of 207.8 mg and a diameter of 7 mmwere prepared to obtain tablets with a target content corresponding to 5mg of the free base.

Example 15i

2290.1 g of the hydrobromide salt, 17568 g anhydrous calcium hydrogenphosphate and 8783 g of corn starch and 1510 g copovidone were mixed for3 minutes in an Aeromatic-Fielder PMA100 high-shear mixer at an impellerspeed of 200 rpm. Next, 5130 g water was added during the course of 2minutes at an impeller speed of 150 rpm. Massing was performed for 15minutes and the resulting granules were passed through a cone milloperating at about 2700 rpm with a screen, size 9.525 mm. The granuleswere dried and passed through cone mill operating at about 1500 rpm witha screen, size 2.388 mm. 28747 g of the resulting granules were mixedwith 11250 g microcrystalline cellulose, 1350 g sodium starch glycolate(type A) and 1800 g talc. Following lubrication of the blend by mixingwith 450 g magnesium stearate the powder blend was transferred to atablet press. Tablets having a target core weight of 125 mg and adiameter of 6 mm were prepared to obtain tablets with a target contentof the hydrobromide salt corresponding to 5 mg of the free base. Inaddition, tablets having a target core weight of 250 mg and a diameterof 8 mm were prepared to obtain tablets with a target content of thehydrobromide salt corresponding to 10 mg of the free base.

Example 16 Pain Effects in the Mouse Intradermal Formalin Test

In this model, mice receive an injection of formalin (4.5%, 20 μl) intothe left hind paw. The irritation caused by the formalin injectionelicits a characteristic biphasic behavioural response, as quantified bythe amount of time spent licking the injured paw. The first phase (˜0-10minutes) represents direct chemical irritation and nociception, whereasthe second (˜20-30 minutes) is thought to represent pain of neuropathicorigin. The two phases are separated by a quiescent period in whichbehaviour returns to normal. The effectiveness of test compounds toreduce the painful stimuli is assessed by counting the amount of timespent licking the injured paw in the two phases.

Compounds of the present invention showed a significant reduction insecond phase pain scores (FIG. 18 b), indicating efficacy against painof neuropathic origin. Furthermore, the compounds of the presentinvention showed a significant reduction in the first phase scores (FIG.18 a), indicating a more analgesic action at the highest dose. Insummary, these results indicate that compounds of the present inventionare likely to be effective in the treatment of pain disorders.

Example 17

20 g 2-bromoiodobenzene (71 mmol) and 9.8 g 2,4-dimethylthiophenol (71mmol) were dissolved in 100 ml toluene. The solution was purged withnitrogen before 324 mg Pd₂dba₃ (0.35 mmol; 1 mol-%) and 381 mg DPEPhos(0.71 mmol; 1 mol-%). The reaction mixture was stirred 5 min duringwhich the colour changes from dark-red to orange. Addition of 8.7 gKOBu^(t) (78 mmol) took place and a heterogeneous mixture was formedinstantly. The suspension was heated to 100° C. under nitrogen. After 1hour the mixture was cooled to 0° C. and stirred for 2 hours beforefiltering the mixture though a pad of celite. The filter cake was washedwith 2×50 ml toluene and the combined filtrates evaporated to 21 g of aorange-reddish oil (99% yield) that proved >96% pure on HPLC and GC-MS.

Example 18

500 ml toluene was placed in a 1 L three necked round bottle with amechanical stirrer and added 203 mg Pddba₂ (0.35 mmol; 0.1 mol-%) and760 mg DPEPhos (1.5 mmol; 0.4 mol-%). The dark-red solution was purgedwith nitrogen for 5 minutes before addition of 100 g 2-bromoiodobenzene(353 mmol) and 48.9 g 2,4-dimethylthiophenol (353 mmol) took place.Addition of 43.6 g KOBu^(t) (389 mmol) caused an exothermic reactionincreasing the temperature from 20° C. to 36° C. simultaneously with theformation of heterogeneous mixture. The suspension was heated to 100° C.under nitrogen. After 7 hours the mixture was cooled to 0° C. andstirred for 2 hours before filtering the mixture though a pad of celite.The filter cake was washed with 2×200 ml toluene and the combinedfiltrates were evaporated to 104 g of an orange oil (105% yield) thatproved 97% pure on HPLC and NMR conforms to desired structure. The oilsolidified during standing at room temperature.

Example 19

A solution of 10 gram 1-(2-Bromo-phenylsulfanyl)-2,4-dimethyl-benzene(34 mmol) in 50 ml dry toluene was added 7 gram boc-piperazine (38mmol), degassed with nitrogen for 5 minutes, added 312 mg Pd₂dba₃ (2mol-%) and 637 mg rac-BINAP (3 mol-%), degassed for another 5 minutesbefore adding 3.9 gram Bu^(t)ONa (41 mmol) and heated to 80° C. for 15hours. The reaction mixture was cooled to RT and extracted twice with 20ml 15% brine, dried over Na₂SO₄ added charcoal, refluxed for 15 minutes,filtered though celite and evaporated to 14.2 gram of brownish oil(4-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-BOC-piperazine) having apurity of 95% determined by NMR. The crude oil was dissolved in 200 mlMeOH and 20 ml 6M HCl (aq.) and refluxed for 1 hour after which HPLCshowed full deprotection. After cooling to RT the methanol was removedby vacuum on a rotary-evaporator, 20 ml conc. NaOH (pH was measured to13-14) was added after which the mixture was stirred 15 minutes with 100ml EtOAc. The organic phase was collected and extracted twice with 30 ml15% brine, dried over Na₂SO₄ and added 5.2 g fumaric acid (44 mmol) in30 ml MeOH. During heating to reflux a homogenous solution forms fromwhich a rapid precipitation takes place either during further heating orupon cooling. The precipitate was collected, washed with 20 ml EtOAc and20 ml acetone, dried in vacuum giving 9.3 gram of1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine fumarate (22 mmol)as a white powder in 66% overall yield having a purity of 99.5% byLC-MS.

Example 20

100 g 1,2-dibromobenzene (424 mmol) and 58.6 g 2,4-dimethylthiophenol(424 mmol) are dissolved in 800 ml toluene. The solution is purged withnitrogen before 4.6 g Pddba₂ (8 mmol; 2 mol-%) and 13.1 g rac-BINAP (21mmol; 5 mol-%). The reaction mixture is stirred 5 min during which thecolour changes from dark-red to orange. Addition of 61 g NaOBu^(t) (636mmol) and 200 ml toluene took place and a heterogeneous mixture wasformed instantly. The suspension was heated to 80° C. under nitrogen.After 10 hours the mixture is cooled to 60° C. before adding a slurry of102.6 g boc-piperazine (551 mmol) and another 61 g NaOBu^(t) (636 mmol)in 500 ml toluene. The reaction mixture was purged with nitrogen beforeadding another portion of 4.6 g Pddba₂ (8 mmol; 2 mol-%) and 13.1 grac-BINAP (21 mmol; 5 mol-%). The mixture was heated to reflux this time(110° C.) for another 6 hours or until HPLC shows full conversion. Thereaction mixture was cooled on ice for 2 hours before filtering themixture though a pad of celite. The filter cake is washed with 2×200 mltoluene and the combined filtrates evaporated to 242 g of red oil. Theoil was dissolved in 1000 ml MeOH and slowly added 115 ml 48-wt % HBr(aq.) followed by heating to reflux for 2 hours after which fulldeprotection was detected by HPLC. The mixture was cooled, added 1000 mlEtOAc and the MeOH was removed by evaporation. Addition of 1000 ml Et₂Ocaused a precipitation. Stirring was continued at room temperature for 2hours before leaving the slurry in the freezer overnight (−18° C.).Filtration and washing twice with 200 ml Et₂O produced 172 g brownishsolid after drying in vacuum at 40° C. The brownish solid was treatedwith 1500 ml boiling H₂O for 1 hour before cooled to room temperaturefor another 2 hours. Filtering and drying in vacuum at 40° C. overnightproduced 98 g of 4-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazinehydrobromide (61%).

Example 21

102 g 2-bromo-iodobenzene (362 mmol) and 50 g 2,4-dimethylthiophenol(362 mmol) are dissolved in 1000 ml toluene. To this solution was added81 g BOC-piperazine (434 mmol) followed by 2.08 g Pddba₂ (1 mol %) and4.51 g rac-BINAP (2 mol %). The mixture was purged with nitrogen for 5minutes before adding a slurry of 87 g NaOBu^(t) (905 mmol) in 300 mltoluene. The suspension was heated to 100° C. under nitrogen overnight.A GCMS analysis showed full conversion into the intermediate product(1-(2-Bromo-phenylsulfanyl)-2,4-dimethyl-benzene) and the temperaturewas increased to reflux (120° C.) for another 24 hours. A HPLC analysisshowed full conversion into the intermediate(1-BOC-4-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine). Thereaction mixture was cooled on ice for one hour before filtering themixture. The filter cake is washed with 2×200 ml toluene and to thecombined filtrates was added 80 ml 48-wt % HBr (aq.) followed by heatingto reflux for 18 hours after which full deprotection was detected byHPLC. The mixture was cooled on ice for 2 hours and filtrated. Thebrownish solid was dissolved in 1000 ml boiling H₂O for 1 hour togetherwith activated charcoal (25 g), filtered while hot and left to cool. Theprecipitate was collected by filtration and drying in vacuum at 40° C.overnight produced 49 g of4-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide (36%)as a white solid.

Example 22

500 ml toluene was placed in a 1 L three-necked round bottle with amechanical stirrer and added 809 mg Pd₂dba₃ (0.88 mmol; 0.5 mol-%) and952 mg DPEPhos (1.77 mmol; 0.5 mol-%). The dark-red solution was purgedwith nitrogen for 5 minutes before addition of 100 g 2-bromoiodobenzene(353 mmol) and 48.9 g 2,4-dimethylthiophenol (353 mmol) took place.Addition of 43.6 g KOBu^(t) (389 mmol) caused an exothermic reactionincreasing the temperature from 20° C. to 42° C. simultaneously with theformation of a heterogeneous mixture and the colour changed fromdark-red into orange/brownish. The suspension was heated to 100° C.under nitrogen. After only 20 minutes a HPLC showed full conversion into1-(2-Bromo-phenylsulfanyl)-2,4-dimethyl-benzene. The mixture was cooledto 40° C., added 600 ml 15-wt % NaCl and stirred for 5 minutes. Theorganic phase was separated and the aqueous phase was washed with 2×100ml toluene. The combined organic phases were washed with 100 ml 2M HCl(aq.), 100 ml 15-wt % NaCl, dried over Na₂SO₄, refluxed for 15 minuteswith activated charcoal (10 g), filtered twice and evaporated to 107.3 gorange-red oil (103%) that was found to be 98% pure by HPLC.

A solution of 90 gram of the orange-red oil (307 mmol) in 500 ml drytoluene was added 57 gram boc-piperazine (307 mmol), degassed withnitrogen for 5 minutes, added 1.4 g Pd₂dba₃ (1.53 mmol; 0.5 mol-%) and2.9 g rac-BINAP (4.6 mmol; 1.5 mol-%), degassed for another 2 minutesbefore adding 35.4 gram Bu^(t)ONa (368 mmol) and heated to 80° C. for 18hours. HPLC showed full conversion and the reaction mixture was cooledto RT, filtered and the filter cake was washed with 2×100 ml toluene.The combined filtrates was extracted twice with 2×150 ml 15-wt % NaCl,dried over Na₂SO₄, added charcoal, refluxed for 30 minutes, filteredtwice and evaporated to 140.7 gram of brownish oil(4-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-BOC-piperazine). The crudeoil was dissolved in 300 ml MeOH and 200 ml 6M HCl (aq.) and refluxedfor 1 hour after which HPLC showed full deprotection. After cooling toRT the methanol was removed by vacuum on a rotary-evaporator, 200 mlconc. NaOH (pH was measured to 13-14) was added after which the mixturewas stirred 15 minutes with 1000 ml EtOAc. The organic phase wascollected and extracted with 300 ml 15-wt % brine, dried over Na₂SO₄ andadded to a solution of 46.3 g fumaric acid (399 mmol) in 300 ml MeOH.The mixture was heated to reflux, cooled to room temperature and thenleft in the freezer overnight (−18° C.). The precipitate was collected,washed with 100 ml EtOAc and 100 ml acetone, dried in vacuum (50° C.)producing 103.2 g of1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine fumarate (249mmol) as a white powder in 81% overall yield having a purity of 99% byLC-MS. The fumarate was transfer into the free base(1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine) usingEtOAc/H₂O/conc. NaOH, the organic phase was washed with brine, driedusing Na₂SO₄, filtered and to the filtrate was added 34 ml 48-wt % HBr(aq.) causing a precipitation of a white solid. The solid was collected,treated with 1000 ml boiling H₂O, which upon cooling to room temperatureformed a slurry. The final product(1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide) wascollected by filtration and dried in vacuum (50° C.) producing 83 g ofwhite powder (71% yield overall), CHN (teo.) 56.99; 6.11; 7.39; CHN(found) 57.11; 6.15; 7.35.

Example 23

815 g NaOBu^(t) (8.48 mol), 844 g Piperazine (9.8 mol), 6.6 g Pd(dba)₂(11.48 mmol) and 13.6 g rac-BINAP (21.84 mmol) were stirred with 4 Ltoluene for 50 minutes. 840 g 2-bromo-iodobenzene (2.97 mol) was thenadded along with 1.5 L Toluene and stirring continued for 30 min. 390.8g 2,4-dimethylthiophenol (2.83 mol) was finally added with 1.5 Ltoluene. The suspension was heated to reflux and reflux continued for 5hours. The reaction mixture was cooled down over night. 2 L water wasadded and stirred for 1 hour before the mixture was filtrated throughfilter aid. The filtrate was then washed with 3×1 L brine. The combinedwater phases were then extracted with 600 ml toluene. The combinedtoluene phases were then heated to 70° C. followed by addition of 329.2ml 48-wt % HBr (aq.) and 164.6 ml water. The mixture was cooled to roomtemperature over night. The final product(1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide) wascollected by filtration and dried in vacuum (60° C.) producing 895 g(84% yield).

Example 24

40.76 g NaOBu^(t) (424.1 mmol), 0.33 g Pddba₂ (0.57 mmol) and 0.68 grac-BINAP (1.09 mmol) were stirred with 200 ml toluene. 42 g2-bromo-iodobenzene (362 mmol) and 19.54 g 2,4-dimethylthiophenol (362mmol) were added with 50 ml toluene. The suspension was heated to refluxand reflux continued over night. A HPLC analysis showed full conversioninto the intermediate product(1-(2-Bromo-phenylsulfanyl)-2,4-dimethyl-benzene). The reaction mixturewas cooled to RT and filtered through filter aid. The filtrate was addedto a mixture of 40.76 g NaOBu^(t) (424.1 mmol), 42.2 g piperazine (489.9mmol), 0.33 g Pddba₂ (0.57 mmol) and 0.68 g rac-BINAP (1.09 mmol) andheated to reflux for 2 hours. The reaction mixture was cooled down overnight. 100 ml water was added and the water phase separated off. Theorganic phase was filtered through filter aid and the filtrate was thenwashed with 3×80 ml brine. The combined water phases were then extractedwith 50 ml toluene. The combined toluene phases were then heated to 70°C. and followed by addition of 16.5 ml 48-wt % HBr (aq.) and 8.25 mlwater. The mixture was cooled to room temperature over night. The finalproduct (1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazinehydrobromide) was collected by filtration and dried in vacuum (60° C.)producing 40.18 g of off-white powder (75% yield)

Example 25

40.76 g NaOBu^(t) (424.1 mmol), 0.33 g Pddba₂ (0.57 mmol) and 0.68 grac-BINAP (1.09 mmol) were stirred with 200 ml toluene. 42 g2-bromo-iodobenzene (148.5 mmol) and 19.54 g 2,4-dimethylthiophenol(141.4 mmol) was added with 50 ml toluene. The suspension was heated toreflux and reflux continued over night. A HPLC analysis showed fullconversion into the intermediate product(1-(2-Bromo-phenylsulfanyl)-2,4-dimethyl-benzene). Reaction cooled to50° C. and 42.2 g piperazine (489.9 mmol) was added along with 100 mltoluene. The mixture was heated to reflux for 4 hours. The reactionmixture was cooled to RT over night. 100 ml water was added and thereaction mixture was filtered through filter aid. The filter cake wasthen washed with 50 ml toluene.

The water phase was separated off and the organic phase was then washedwith 3×25 ml brine and 25 ml water. The combined water phases were thenextracted with 30 ml toluene. The combined toluene phases was thenheated to 70° C. and followed by addition of 16.46 ml 48-wt % HBr (aq.)and 8.23 ml water. The mixture was cooled to room temperature overnight. The final product(1-[2-(2,4-Dimethyl-phenylsulfanyl)-phenyl]-piperazine hydrobromide) wascollected by filtration and dried in vacuum (60° C.) producing 46.8 g(87% yield).

Example 26 Effects on Extracellular Levels of Acetylcholine in the Brainof Freely Moving Rats

Methods

The animals were administered1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piparazine, HBr salt.

Animals

Male Sprague-Dawley rats, initially weighing 275-300 g, were used. Theanimals were housed under a 12-hr light/dark cycle under controlledconditions for regular in-door temperature (21±2° C.) and humidity(55±5%) with food and tap water available ad libitum.

Surgery and Microdialysis Experiments

Rats were anaesthetised with hypnorm/dormicum (2 ml/kg) andintracerebral guide cannulas (CMA/12) were stereotaxically implantedinto the brain, aiming at positioning the dialysis probe tip in theventral hippocampus (co-ordinates: 5.6 mm posterior to bregma,lateral—5.0 mm, 7.0 mm ventral to dura) or in the prefrontal cortex(co-ordinates: 3.2 mm anterior to bregma; lateral, 0.8 mm; 4.0 mmventral to dura). Anchor screws and acrylic cement were used forfixation of the guide cannulas. The body temperature of the animals wasmonitored by rectal probe and maintained at 37° C. The rats were allowedto recover from surgery for 2 days, housed singly in cages. On the dayof the experiment a microdialysis probe (CMA/12, 0.5 mm diameter, 3 mmlength) was inserted through the guide cannula.

The probes were connected via a dual channel swivel to a microinjectionpump. Perfusion of the microdialysis probe with filtered Ringer solution(145 mm NaCl, 3 mM KCl, 1 mM MgCl₂, 1.2 mM CaCl₂ containing 0.5 μMneostigmine) was begun shortly before insertion of the probe into thebrain and continued for the duration of the experiment at a constantflow rate of 1 μl/min. After 180 min of stabilisation, the experimentswere initiated. Dialysates were collected every 20 min. After theexperiments the animals were sacrificed, their brains removed, frozenand sliced for probe placement verification.

The compound dissolved in 10% HPbetaCD and injected subcutaneously(2.5-10 mg/kg). Doses are expressed as mg salt/kg body weight. Thecompound was administered in a volume of 2.5 ml/kg.

Analysis of Dialysate Acetylcholine

Concentration of acetylcholine (ACh) in the dialysates was analysed bymeans of HPLC with electrochemical detection using a mobile phaseconsisting of 100 mM disodium hydrogenphosphate, 2.0 mM octane sulfonicacid, 0.5 mM tetramethyl-ammonium chloride and 0.005% MB (ESA), pH 8.0.A pre-column enzyme reactor (ESA) containing immobilised choline oxidaseeliminated choline from the injected sample (10 μl) prior to separationof ACh on the analytical column (ESA ACH-250); flow rate 0.35 ml/min,temperature: 35° C. After the analytical column the sample passedthrough a post-column solid phase reactor (ESA) containing immobilisedacetylcholineesterase and choline oxidase. The latter reactor convertedACh to choline and subsequently choline to betaine and H₂O₂. The latterwas detected electrochemical by using a platinum electrode (Analyticalcell: ESA, model 5040).

Data Presentation

In single injection experiments the mean value of 3 consecutive AChsamples immediately preceding compound administration served as thebasal level for each experiment and data were converted to percentage ofbasal (mean basal pre-injection values normalized to 100%).

Results

The compound significantly increased extra-cellular levels of ACh in therat prefrontal cortex and the ventral hippocampus—see FIGS. 19 a and 19b.

Example 27 Contextual Fear Conditioning in Rats

The compound administered in the present experiment was1-[2-(2,4-dimethylphenylsulfanyl)phenyl]piperazine HBr salt.

We have studied the effect of the compound on acquisition, consolidationand recall of contextual fear conditioning in rats. In the fearconditioning paradigm animals learn to associate a neutral environment(context, the training chamber, CS) with an aversive experience (anelectrical foot-shock, US). During re-exposure to the training chamber,animals express a freezing behaviour, which is taken as a direct measureof the fear-related memory [Pavlov J. Biol. Sci., 15, 177-182, 1980].The neuroanatomy of contextual fear conditioning has been thoroughlyinvestigated and several studies have demonstrated that the hippocampusand amygdala are necessary for the formation of this memory[Hippocampus, 11, 8-17, 2001; J. Neurosci., 19, 1106-1114, 1999; Behav.Neurosci., 106, 274-285, 1992].

Animals and Drugs

Adult male Sprague-Dawley rats (weighing 250-300 g at time of training)from Charles River Laboratories, housed two per cage under a 12 hlight/dark cycle, were used. Food and water were available ad libitum.Rats were used 1 week after arrival. The compound was dissolved in 10%HPbetaCD and injected subcutaneously. The drug was administered in avolume of 2.5 ml/kg.

Apparatus

Training and testing were conducted in a soundproof chamber (30×20×40cm) housed in an isolated room and connected to a ventilation system.Illumination was provided by a white light (60 Watt). The floor of thechamber consisted of a metal grid attached to an electric shockgenerator. Prior to training and testing, the chamber was cleaned with a70% ethanol solution. A video camera allowed for behaviouralobservations and recording of the training session for off-lineanalysis.

Acquisition and Retention Test

During the acquisition animals were allowed to freely explore the novelenvironment for a 1 min habituation period, which co-terminated with oneinescapable foot-shock (unconditioned stimulus, US) through theelectrifiable grid floor. The foot shock had a duration of 2 s and anintensity of 0.75 mA. Animals remained in the conditioning chamber foranother 60 s after the US. Freezing behaviour was scored during thefirst 58 s (pre-shock acquisition; experimenter blinded to groups) todetermine baseline-freezing responses to the context. At the end of theacquisition animals were gently removed and placed into their homecages. After 24 h the same animals were reintroduced into the trainingcontext (fear conditioning chamber) and a 2 min retention test wasperformed. During this period no foot shocks were applied. Freezingbehaviour was scored during the whole test period with the experimenterblinded to groups and presented as percent of total test period.

Results and Discussion

Effect of the Compound on Contextual Fear Cognition in Rats

The effect of the compound on contextual fear conditioning in rats wasstudied (i) on acquisition (drug applied before acquisition, FIG. 20),(ii) on memory recall (drug applied before test, FIG. 21) and (iii) onconsolidation (drug applied immediately after the acquisition, FIG. 22).In the first set of experiments, the compound (1, 5 and 10 mg/kg) wasadministered 1 h prior to the acquisition session. FIG. 20 depicts theacquisition of freezing behavior during training (58 s prior to the foodshock) and the retention test 24 after. The following findings wereobserved:

-   -   The compound does not affect baseline freezing behaviour before        the presentation of the foot shock at any dose tested.    -   The compound at 5 mg/kg has a tendency to increase the time        spent freezing during the retention test, 24 h after the        acquisition (39.24±13.76%, n=6, versus 24.30±4.40%, n=16, in the        vehicle-treated animals).    -   The compound at 10 mg/kg significantly increases the time spent        freezing during the retention test, 24 h after the acquisition        (52.15±5.68%, n=10, versus 24.30±4.40%, n=16, in the        vehicle-treated animals, p<0.01).

The fear conditioning model, as described in FIG. 20, is a standardprocedure described in the literature for the investigation of learningand memory. In order to further elucidate the acute effects of this drugon memory recall, the compound (5, 10 and 20 mg/kg) was applied 1 hprior to the retention test. It was observed that the compound inhibitsthe expression of freezing behaviour at 5 mg/kg during the memory test(12.86±3.57%, n=9, versus 33.61±4.29%, n=13, in the vehicle-treatedanimals, p<0.05) (FIG. 21).

As described above, the compound by itself does not affect baselinefreezing behaviour before the onset of US (FIG. 20), thus the mostplausible hypothesis is that the observed effect in FIG. 21 is due to ananxiolytic effect. The conditioned memory is assessed via freezingbehaviour, a response that is reduced by compounds with potentialanxiolytic effects. This experiment demonstrates that the compound givenacutely before memory recall has anxiolytic efficacy, it is thereforeunlikely that increased freezing shown in FIG. 20 is due to ananxiogenic effect of the compound.

In order to strengthen that the compound is not anxiogenic but bearspro-cognitive potential, the compound was administered at 5, 10 and 20mg/kg after the acquisition session. Consequently, in this set ofexperiments, the compound was onboard neither during the acquisition northroughout the retention test. Here, it was observed that the compoundat 5 mg/kg significantly enhances the time spent freezing during theretention test, 24 h after the acquisition session (45.58±4.50%, n=8,versus 25.26±3.57%, n=19, in the vehicle-treated animals, p<0.05). Thepercentage of time spent freezing during the context re-exposure hasbeen described as a measure of a fear-related memory [Pavlov J. Biol.Sci, 15, 177-182, 1980], which is enhanced in compound-treated rats whencompared to vehicle-treated animals (FIGS. 20 and 21). Taken together,the data show that the compound enhances contextual memory.

What is claimed is:
 1. A method of treating a disease selected from thegroup consisting of affective disorders, depression, major depressivedisorder, anxiety, general anxiety disorder, social anxiety disorder,obsessive compulsive disorder, panic disorder, and panic attacks, themethod comprising administering a therapeutically effective amount of apharmaceutically acceptable salt of Compound I to a patient in needthereof, wherein the salt of Compound I is crystalline1-[2-(2,4-dimethylphenylsulfanyl)-phenyl]piperazine hydrobromide saltcharacterized by XRPD reflections at 6.89, 9.73, 13.78 and 14.64+/−0.10°2θ.
 2. The method of claim 1, wherein the hydrobromide salt ischaracterized by an XRPD as shown in FIG.
 3. 3. The method of claim 1,wherein the hydrobromide salt has a particle size distributioncorresponding to: D98%: 650-680 μm; D50%: 230-250 μm; and D5%: 40-60 μm;D98%: 370-390 μm; d50%: 100-120 μm; and D5%: 5-15 μm; D98%: 100-125 μm;D50%: 15-25 μm; and D5%: 1-3 μm; or D98%: 50-70 μm; D50%: 3-7 μm; andD5%: 0.5-2 μm.
 4. The method of claim 1, wherein the disease is majordepressive disorder.
 5. A method of treating a disease selected from thegroup consisting of affective disorders, depression, major depressivedisorder, anxiety, general anxiety disorder, social anxiety disorder,obsessive compulsive disorder, panic disorder, and panic attacks, themethod comprising administering a therapeutically effective amount ofCompound I or a pharmaceutically acceptable salt thereof to a patient inneed thereof, wherein Compound I is1-[2-(2,4-dimethylphenylsulfanyl)-phenyl]piperazine and Compound I or apharmaceutically acceptable salt thereof is in a crystalline formcharacterized by an XRPD pattern as shown in any of FIGS. 1-17.
 6. Themethod of claim 5, wherein Compound I or a pharmaceutically acceptablesalt thereof is a hydrobromide salt of Compound I having a particle sizedistribution corresponding to: D98%: 650-680 μm; D50%: 230-250 μm; andD5%: 40-60 μm; D98%: 370-390 μm; d50%: 100-120 μm; and D5%: 5-15 μm;D98%: 100-125 μm; D50%: 15-25 μm; and D5%: 1-3 μm; or D98%: 50-70 μm;D50%: 3-7 μm; and D5%: 0.5-2 μm.
 7. The method of claim 5, wherein thedisease is major depressive disorder.